Ricin-like toxins for treatment of cancer

ABSTRACT

The present invention provides a protein having chain of a ricin-like toxin, a B chain of a ricin-like toxin and a novel heterologous linker amino acid sequence, linking the A and B chains. The linker sequence contains a cleavage recognition site for a specific protease such as those found in inflammatory cells and cancer cells. The invention also relates to a nucleic acid molecule encoding the protein and to expression vectors incorporating the nucleic acid molecule. Also provided is a method of inhibiting or destroying cells having a specific protease, such as cancer cells or inflammatory cells utilizing the nucleic acid molecules and proteins of the invention and pharmaceutical compositions for treating human inflammation and cancer.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/089,058 (now allowed) filed Sep. 19, 2002 which is a continuation ofPCT/CA00/01162 filed Oct. 4, 2000 (which designated the U.S.) whichclaims the benefit of U.S. Provisional application No. 60/157,807 filedOct. 4, 1999 (now abandoned). This application also claims benefit ofU.S. Provisional application No. 60/197,409 filed Apr. 14, 2000 (nowabandoned). All of the prior applications are incorporated herein intheir entirety.

FIELD OF THE INVENTION

The invention relates to proteins useful as therapeutics againstunhealthy cells such as those which occur in inflammation and cancer.The proteins contain A and B chains of a ricin-like toxin linked by anovel linker sequence that is specifically cleaved and activated byproteases specific to cancer.

BACKGROUND OF THE INVENTION

Bacteria and plants are known to produce cytotoxic proteins which mayconsist of one, two or several polypeptides or subunits. Those proteinshaving a single subunit may be loosely classified as Type I proteins.Many of the cytotoxins which have evolved two subunit structures arereferred to as Type II proteins (Saelinger, C. B. in Trafficking ofBacterial Toxins (eds. Saelinger, C. B.) 1-13 (CRC Press Inc., BocaRaton, Fla., 1990). One subunit, the A chain, possesses the toxicactivity whereas the second subunit, the B chain, binds cell surfacesand mediates entry of the toxin into a target cell. A subset of thesetoxins kill target cells by inhibiting protein biosynthesis. Forexample, bacterial toxins such as diphtheria toxin or Pseudomonasexotoxin inhibit protein synthesis by inactivating elongation factor 2.Plant toxins such as ricin, abrin, and bacterial toxin Shiga toxin,inhibit protein synthesis by directly inactivating the ribosomes(Olsnes, S. & Phil, A. in Molecular action of toxins and viruses (eds.Cohen, P. & vanHeyningen, S.) 51-105 Elsevier Biomedical Press,Amsterdam, 1982).

Ricin, derived from the seeds of Ricinus communis (castor oil plant),may be the most potent of the plant toxins. It is estimated that asingle ricin A chain is able to inactivate ribosomes at a rate of 1500ribosomes/minute. Consequently, a single molecule of ricin is enough tokill a cell (Olsnes, S. & Phil, A. in Molecular action of toxins andviruses (eds. Cohen, P. & vanHeyningen, S.) (Elsevier Biomedical Press,Amsterdam, 1982). The ricin toxin is a glycosylated heterodimerconsisting of A and B chains with molecular masses of 30,625 Da and31,431 Da linked by a disulphide bond. The A chain of ricin has anN-glycosidase activity and catalyzes the excision of a specific adenineresidue from the 28S rRNA of eukaryotic ribosomes (Endo, Y. & Tsurugi,K. J., Biol. Chem. 262:8128 (1987)). The B chain of ricin, although nottoxic in itself, promotes the toxicity of the A chain by binding togalactose residues on the surface of eukaryotic cells and stimulatingreceptor-mediated endocytosis of the toxin molecule (Simmons et al.,Biol. Chem. 261:7912 (1986)). Once the toxin molecule consisting of theA and B chains is internalized into the cell via clathrin-dependent orindependent mechanisms, the greater reduction potential within the cellinduces a release of the active A chain, eliciting its inhibitory effecton protein synthesis and its cytotoxicity (Emmanuel, F. et al., Anal.Biochem. 173: 134-141 (1988); Blum, J. S. et al., J. Biol. Chem. 266:22091-22095 (1991); Fiani, M. L. et al., Arch. Biochem. Biophys. 307:225-230 (1993)). Empirical evidence suggests that activated toxin (e.g.ricin, shiga toxin and others) in the endosomes is transcytosed throughthe trans-Golgi network to the endoplasmic reticulum by retrogradetransport before the A chain is translocated into the cytoplasm toelicit its action (Sandvig, K. & van Deurs, B., FEBS Lett. 346: 99-102(1994).

Protein toxins are initially produced in an inactive, precursor form.Ricin is initially produced as a single polypeptide (preproricin) withan amino acid N-terminal presequence and 12 amino acid linker betweenthe A and B chains. The pre-sequence is removed during translocation ofthe ricin precursor into the endoplasmic reticulum (Lord, J. M., Eur. J.Biochem. 146:403-409 (1985) and Lord, J. M., Eur. J. Biochem.146:411-416 (1985)). The proricin is then translocated into specializedorganelles called protein bodies where a plant protease cleaves theprotein at a linker region between the A and B chains (Lord, J. M. etal., FASAB journal 8:201-208 (1994)). The two chains, however, remaincovalently attached by an interchain disulfide bond (cysteine 259 in theA chain to cysteine 4 in the B chain) and mature disulfide linked ricinis stored in protein bodies inside the plant cells. The A chain isinactive in proricin (O'Hare, M. et al., FEBS Lett. 273:200-204 (1990))and it is inactive in the disulfide-linked mature ricin (Richardson, P.T. et al., FEBS Lett. 255:15-20 (1989)). The ribosomes of the castorbean plant are themselves susceptible to inactivation by ricin A chain;however, as there is no cell surface galactose to permit B chainrecognition the A chain cannot re-enter the cell. The exact mechanism ofA chain release and activation in target cell cytoplasm is not known(Lord, J. M. et al., FASAB journal 8:201-208 (1994)). However, it isknown that for activation to take place the disulfide bond between the Aand B chains must be reduced and, hence, the linkage between subunitsbroken.

Diphtheria toxin is produced by Corynebacterium diphtheriae as a 535amino acid polypeptide with a molecular weight of approximately 58 kD(Greenfield, L. et al., Proc. Natl. Acad. Sci. USA 80:6853-6857 (1983);Pastan, I. et al., Annu. Rev. Biochem. 61:331-354 (1992); Collier, R. J.& Kandel, J., 1. Biol. Chem. 246:1496-1503 (1971)). It is secreted as asingle-chain polypeptide consisting of 2 functional domains. Similar toproricin, the N-terminal domain (A-chain) contains the cytotoxic moietywhereas the C-terminal domain (B-chain) is responsible for binding tothe cells and facilitates toxin endocytosis. Conversely, the mechanismof cytotoxicity for diphtheria toxin is based on ADP-ribosylation ofEF-2 thereby blocking protein synthesis and producing cell death. The 2functional domains in diphtheria toxin are linked by an arginine-richpeptide sequence as well as a disulphide bond. Once the diphtheria toxinis internalized into the cell, the arginine-rich peptide linker iscleaved by trypsin-like enzymes and the disulphide bond (Cys 186-201) isreduced. The cytotoxic domain is subsequently translocated into thecytosol substantially as described above for ricin and elicits ribosomalinhibition and cytotoxicity.

Pseudomonas exotoxin is also a 66 kD single-chain toxin protein secretedby Pseudomonas aeruginosa with a similar mechanism of cytotoxicity tothat of diphtheria toxin (Pastan, I. et al., Annu. Rev. Biochem.61:331-354 (1992); Ogata, M. et al., J. Biol. Chem. 267:25396 25401(1992); Vagil, M. L. et al., Infect. Immunol. 16:353-361 (1977)).Pseudomonas exotoxin consists of 3 conjoint functional domains. Thefirst domain I (amino acids 1-252) is responsible for cell binding andtoxin endocytosis, a second domain II (amino acids 253-364) isresponsible for toxin translocation from the endocytic vesicle to thecytosol, and a third domain III (amino acids 400-613) is responsible forprotein synthesis inhibition and cytotoxicity. After Pseudomonasexotoxin enters the cell, the liberation of the cytotoxic domain iseffected by both proteolytic cleavage of a polypeptide sequence in thesecond domain (near Arg 279) and the reduction of the disulphide bond(Cys 265-287) in the endocytic vesicles. In essence, the overall pathwayto cytotoxicity is analogous to diphtheria toxin with the exception thatthe toxin translocation domain in Pseudomonas exotoxin is structurallydistinct.

Class 2 ribosomal inhibitory proteins (RIP-2) constitute other toxinspossessing distinct functional domains for cytotoxicity and cellbinding/toxin translocation which include abrin, modeccin, volkensin,(Sandvig, K. et al., Biochem. Soc. Trans. 21:707-711 (1993)) and mistletoe lectin (viscumin) (Olsnes, S. & Phil, A. in Molecular action oftoxins and viruses (eds. Cohen, P. & vanHeyningen, S.) 51-105 ElsevierBiomedical Press, Amsterdam, 1982; Fodstad, et al. Canc. Res. 44: 862(1984)). Some toxins such as Shiga toxin and cholera toxin also havemultiple polypeptide chains responsible for receptor binding andendocytosis.

The ricin gene has been cloned and sequenced, and the X-ray crystalstructures of the A and B chains have been described (Rutenber, E. etal. Proteins 10:240-250 (1991); Weston et al., Mol. Bio. 244:410-422,1994; Lamb and Lord, Eur. J. Biochem. 14:265 (1985); Halling, K. et al.Nucleic Acids Res. 13:8019 (1985)). Similarly, the genes for diptheriatoxin and Pseudomonas exotoxin have been cloned and sequenced, and the3-dimensional structures of the toxin proteins have been elucidated anddescribed (Columblatti, M. et al., J. Biol. Chem. 261:3030-3035 (1986);Allured, V. S. et al., Proc. Natl. Acad. Sci. USA 83:1320-1324 (1986);Gray, G. L. et al., Proc. Natl. Acad. Sci. USA 81:2645-2649 (1984);Greenfield, L. et al., Proc, Natl. Acad. Sci. USA 80:6853-6857 (1983);Collier, R. J. et al., J. Biol. Chem. 257:5283-5285 (1982)).

The potential of bacterial and plant toxins for inhibiting mammalianretroviruses, particularly acquired immunodeficiency syndrome (AIDS),has been investigated. Bacterial toxins such as Pseudomonas exotoxin andsubunit A of diphtheria toxin; dual chain ribosomal inhibitory planttoxins such as ricin, and single chain ribosomal inhibitory proteinssuch as trichosanthin and pokeweed antiviral protein have been used forthe elimination of HIV infected cells (Olson et al., AIDS Res. and HumanRetroviruses 7:1025-1030 (1991)). The high toxicity of these toxins formammalian cells, combined with a lack of specificity of action poses amajor problem to the development of pharmaceuticals incorporating thetoxins, such as immunotoxins.

Due to their extreme toxicity there has been much interest in makingricin-based immunotoxins as therapeutic agents for specificallydestroying or inhibiting infected or tumourous cells or tissues (Vitettaet al., Science 238:1098-1104 (1987)). An immunotoxin is a conjugate ofa specific cell binding component, such as a monoclonal antibody orgrowth factor and the toxin in which the two protein components arecovalently linked. Generally, the components are chemically coupled.However, the linkage may also be a peptide or disulfide bond. Theantibody directs the toxin to cell types presenting a specific antigenthereby providing a specificity of action not possible with the naturaltoxin. Immunotoxins have been made both with the entire ricin molecule(i.e. both chains) and with the ricin A chain alone (Spooner et al.,Mol. Immunol. 31:117-125, (1994)).

Immunotoxins made with the ricin dimer (IT-Rs) are more potent toxinsthan those made with only the A chain (IT-As). The increased toxicity ofIT-Rs is thought to be attributed to the dual role of the B chains inbinding to the cell surface and in translocating the A chain to thecytosolic compartment of the target cell (Vitetta et al., Science238:1098-1104 (1987); Vitetta & Thorpe, Seminars in Cell Biology 2:47-58(1991)). However, the presence of the B chain in these conjugates alsopromotes the entry of the immunotoxin into nontarget cells. Even smallamounts of B chain may override the specificity of the cell-bindingcomponent as the B chain will bind nonspecifically to galactoseassociated with N-linked carbohydrates, which is present on most cells.IT-As are more specific and safer to use than IT-Rs. However, in theabsence of the B chain the A chain has greatly reduced toxicity. Due tothe reduced potency of IT-As as compared to IT-Rs, large doses of IT-Asmust be administered to patients. The large doses frequently causeimmune responses and production of neutralizing antibodies in patients(Vitetta et al., Science 238:1098-1104 (1987)). IT-As and IT-Rs bothsuffer from reduced toxicity as the A chain is not released from theconjugate into the target cell cytoplasm.

A number of immunotoxins have been designed to recognize antigens on thesurfaces of tumour cells and cells of the immune system (Pastan et al.,Annals New York Academy of Sciences 758:345-353 (1995)). A major problemwith the use of such immunotoxins is that the antibody component is itsonly targeting mechanism and the target antigen is often found onnon-target cells (Vitetta et al., Immunology Today 14:252-259 (1993)).Also, the preparation of a suitable specific cell binding component maybe problematic. For example, antigens specific for the target cell maynot be available and many potential target cells and infective organismscan alter their antigenic make up rapidly to avoid immune recognition.In view of the extreme toxicity of proteins such as ricin, the lack ofspecificity of the immunotoxins may severely limit their usefulness astherapeutics for the treatment of cancer and infectious diseases.

The insertion of intramolecular protease cleavage sites between thecytotoxic and cell-binding components of a toxin can mimic the way thatthe natural toxin is activated. European patent application no. 466,222describes the use of maize-derived pro-proteins which can be convertedinto active form by cleavage with extracellular blood enzymes such asfactor, Xa, thrombin or collagenase. Garred, 0. et al. (J. Biol. Chem.270:10817-10821 (1995)) documented the use of a ubiquitouscalcium-dependent serine protease, furin, to activate shiga toxin bycleavage of the trypsin-sensitive linkage between the cytotoxic A-chainand the pentamer of cell-binding B-units. Westby et al. (BioconjugateChem. 3:375-381 (1992)) documented fusion proteins which have a specificcell binding component and proricin with a protease sensitive cleavagesite specific for factor Xa within the linker sequence. O'Hare et al.(FEBS Lett. 273:200-204 (1990)) also described a recombinant fusionprotein of RTA and staphylococcal protein A joined by atrypsin-sensitive cleavage site. In view of the ubiquitous nature of theextracellular proteases utilized in these approaches, such artificialactivation of the toxin precursor or immunotoxin does not confer amechanism for intracellular toxin activation and the problems of targetspecificity and adverse immunological reactions to the cell-bindingcomponent of the immunotoxin remain.

In a variation of the approach of insertion of intramolecular proteasecleavage sites on proteins which combine a binding chain and a toxicchain, Leppla, S. H. et al. (Bacterial Protein Toxins zbl.bakt.suppl.24:431-442 (1994)) suggest the replacement of the native cleavage siteof the protective antigen (PA) produced by Bacillus anthracis with acleavage site that is recognized by cells that contain a particularprotease. PA, recognizes, binds, and thereby assists in theinternalization of lethal factor (U) and edema toxin (ET), also producedby Bacillus anthracis. However, this approach is wholly dependent on theavailability of LF, or ET and PA all being localized to cells whereinthe modified PA can be activated by the specific protease. It does notconfer a mechanism for intracellular toxin activation and presents aproblem of ensuring sufficient quantities of toxin for internalizationin target cells.

The in vitro activation of a Staphylococcus-derived pore forming toxin,α-hemolysin by extracellular tumour-associated proteases has beendocumented (Panchel, R. G. et al., Nature Biotechnology 14:852-857(1996)). Artificial activation of α-hemolysin in vitro by said proteaseswas reported but the actual activity and utility of α-hemolysin in thedestruction of target cells were not demonstrated.

α-Hemolysin does not inhibit protein synthesis but is a heptamerictransmembrane pore which acts as a channel to allow leakage of moleculesup to 3 kD thereby disrupting the ionic balances of the living cell. Theα-hemolysin activation domain is likely located on the outside of thetarget cell (for activation by extracellular proteases). The triggeringmechanism in the disclosed hemolysin precursor does not involve theintracellular proteolytic cleavage of 2 functionally distinct domains.Also, the proteases used for the a-hemolysin activation areubitquitiously secreted extracellular proteases and toxin activationwould not be confined to activation in the vicinity of diseased cells.Such widespread activation of the toxin does not confer targetspecificity and limits the usefulness of said a-hemolysin toxin astherapeutics due to systemic toxicity.

A variety of proteases specifically associated with malignancy have beenidentified and described. For example, cathepsin is a family of serine,cysteine or aspartic endopeptidases and exopeptidases which has beenimplicated to play a primary role in cancer metastasis (Schwartz, M. K.,Clin. Chim. Acta 237:67-78 (1995); Spiess, E. et al., J. Histochem.Cytochem. 42:917-929 (1-994); Scarborough, P. E. et al., Protein Sci.2:264276 (1993); Sloane, B. F. et al., Proc. Natl. Acad. Sci. USA83:2483-2487 (1986); Mikkelsen, T. et al., J. Neurosurge 83:285-290(1995)). Matrix metalloproteinases (MMPs or matrixins) arezinc-dependent proteinases consisting of collagenases, matrilysin,stromelysins, stromelysin-like MMPs, gelatinases, macrophage elastase,membrane-type MMPs (MT-MMPs) (Krane, S. M., Ann, N.Y. Acad. Sci.732:1-10 (1994); Woessner, J. F., Ann, N.Y. Acad. Sci. 732:11-21 (1994);Carvalho, K. et al., Biochem. Biophys. Res., Comm. 191:172-179 (1993);Nakano, A. et al. J. of Neurosurge, 83:298-307 (1995); Peng, K-W, et al.Human Gene Therapy, 8:729-738 (1997); More, D. H. et al. Gynaecologiconcology, 65:78-82 (1997), Ravanti, L., Kahari, V. Intl. J. Mol. Med.6(4):391 (2000)). These proteases are involved in pathological matrixremodeling. Under normal physiological conditions, regulation ofmatrixin activity is effected at the level of gene expression. Enzymaticactivity is also controlled stringently by tissue inhibitors ofmetalloproteinases (TIMPs) (Murphy, G. et al., Ann. N.Y. Acad. Sci.,732:31-41 (1994)). The expression of MMP genes is reported to beactivated in inflammatory disorders (e.g. rheumatoid arthritis) andmalignancy.

The present inventors have prepared novel recombinant toxic proteinswhich are specifically toxic to diseased cells but do not depend fortheir specificity of action on a specific cell binding component. Therecombinant proteins toxins have an A chain of a ricin-like toxin linkedto a B chain by a synthetic linker sequence which may be cleavedspecifically by a protease localised in cells or tissues affected by aspecific disease to liberate the toxic A chain thereby selectivelyinhibiting or destroying the diseased cells or tissues (WO 98/49311published Nov. 5, 1998 which is incorporated herein by reference).

SUMMARY OF THE INVENTION

The present invention relates to novel linker sequences that can be usedto prepare recombinant toxic proteins having an A chain of a ricin-liketoxin linked to a B chain by the linker sequence. The novel linkersequences of the invention are illustrated in FIGS. 1-18.

In one aspect the present invention provides a purified and isolatednucleic acid encoding a linker sequence comprising: the nucleic acidsequence of pAP301 as shown in FIG. 1A; the nucleic acid sequence ofpAP302 as shown in FIG. 2A; the nucleic acid sequence of pAP303 as shownin FIG. 3A; the nucleic acid sequence of pAP304 as shown in FIG. 4A; thenucleic acid sequence of pAP305 as shown in FIG. 5A; the nucleic acidsequence of pAP308 as shown in FIG. 6A; the nucleic acid sequence ofpAP309 as shown in FIG. 7A; the nucleic acid sequence of pAP313 as shownin FIG. 8A; the nucleic acid sequence of pAP314 as shown in FIG. 9A; thenucleic acid sequence of pAP315 as shown in FIG. 10A; the nucleic acidsequence of pAP316 as shown in FIG. 11A; the nucleic acid sequence ofpAP318 as shown in FIG. 12A; the nucleic acid sequence of pAP320 asshown in FIG. 13A; the nucleic acid sequence of pAP321 as shown in FIG.14A; the nucleic acid sequence of pAP322 as shown in FIG. 15A; thenucleic acid sequence of pAP323 as shown in FIG. 16A; the nucleic acidsequence of pAP324 as shown in FIG. 17A; and the nucleic acid sequenceof pAP325 as shown in FIG. 18A.

In another aspect, the present invention provides a purified andisolated nucleic acid encoding a recombinant toxic protein comprising(a) a nucleotide sequence encoding an A chain of a ricin-like toxin, (b)a nucleotide sequence encoding a B chain of a ricin-like toxin and (c) aheterologous linker amino acid sequence, linking the A and B chains. Thelinker sequence is not a native linker sequence of a ricin-like toxin,but rather a synthetic heterologous linker sequence containing acleavage recognition site for a specific protease. The A and or the Bchain may be those of ricin. As used herein “specific protease” means aprotease in any cell wherein there is expression of the protease atlevels greater than those found in a corresponding healthy cell.Examples of a specific protease include MMPs, preferably MMP-2, MMP-9,MMP-14, and MT1-MMPs, and UPA, as well as others found in inflammatorycells and malignant cells. An inflammatory cell includes any cellinvolved in the inflammation process having a specific protease.

The recombinant toxic proteins employing the novel linker sequences ofthe present invention may be used to treat various forms of cells havingspecific proteases such as inflammatory disorders including rheumatoidarthritis, atherosclerotic cells, Crohn's disease, central nervoussystem disease as well as in cancer including, but not limited to, T-and B-cell lymphoproliferative diseases, ovarian cancer, pancreaticcancer, head and neck cancer, squamous cell carcinoma, gastrointestinalcancer, breast cancer, prostate, cancer and non small cell lung cancer.In an embodiment, of the invention the cleavage recognition site of thelinker is the cleavage recognition site for a cancer-associatedprotease.

In particular embodiments, the amino acid sequence of the linkercomprises the sequence of PAP301 shown in FIG. 1C; the sequence ofPAP302 shown in FIG. 2C; the sequence of PAP303 shown in FIG. 3C; thesequence of PAP304 shown in FIG. 4C; the sequence of PAP305 shown inFIG. 5C; the sequence of PAP308 shown in FIG. 6C; the sequence of PAP309shown in FIG. 7C; the sequence of PAP316 shown in FIG. 11C; the sequenceof PAP318 shown in FIG. 12C; the sequence of PAP323 shown in FIG. 16C;the sequence of PAP324 shown in FIG. 17C; and the sequence of PAP325shown in FIG. 18C; all cleaved by MMP-9; the sequence of PAP313 shown inFIG. 8C; the sequence of PAP314 shown in FIG. 9C; the sequence of PAP315shown in FIG. 10C; the sequence of PAP320 shown in FIG. 13C; thesequence of PAP321 shown in FIG. 14C; the sequence of PAP322 shown inFIG. 15C; all cleaved by urokinase-type plasminogen activator.

In a preferred embodiment, the nucleic acid sequences of the recombinanttoxic proteins containing ricin A and B chains with each of the linkersequences are shown in FIGS. 1B, 2B, 3B, 4B, 5B, 6B, 7B, 8B, 9B, 10B,11B, 12B, 13B, 14B, 15B, 16B, 17B and 18B.

The present invention also provides a plasmid incorporating the nucleicacid of the invention. In another embodiment, the present inventionprovides a baculovirus transfer vector incorporating the nucleic acid ofthe invention.

In an aspect, the present invention provides a recombinant proteincomprising an A chain of a ricin-like toxin, a B chain of a ricin-liketoxin and a heterologous linker amino acid sequence, linking the A and Bchains, wherein the linker sequence contains a cleavage recognition sitefor a specific protease. The A and/or the B chain may be those of ricin.

In a further aspect, the present invention provides a recombinantprotein comprising an A chain of a ricin-like toxin, a B chain of aricin-like toxin and a heterologous linker amino acid sequence, linkingthe A and B chains, wherein the linker sequence contains a cleavagerecognition site for a inflammatory disease specific protease. The Aand/or the B chain may be those of ricin. In an embodiment, the cleavagerecognition site is the cleavage recognition site for an inflammationbased protease substantially as described above. In a particularembodiment the inflammation is rheumatoid arthritis, atheroscleroticcells, Crohn's disease, or central nervous system disease.

In a further aspect, the present invention provides a recombinantprotein comprising an A chain of a ricin-like toxin, a B chain of aricin-like toxin and a heterologous linker amino acid sequence, linkingthe A and B chains, wherein the linker sequence contains a cleavagerecognition site for a cancer-specific protease. The A and/or the Bchain may be those of ricin. In an embodiment, the cleavage recognitionsite is the cleavage recognition site for a cancer proteasesubstantially as described above. In a particular embodiment, the canceris T-cell or B-cell lymphoproliferative disease, ovarian cancer,pancreatic cancer, head and neck cancer, squamous cell carcinoma,gastrointestinal cancer, breast cancer, prostate cancer, non small celllung cancer.

In a further aspect, the invention provides a pharmaceutical compositionfor treating a cell, such as an inflammatory cell or cancer cell, havinga specific protease, comprising a recombinant protein of the inventionand a pharmaceutically acceptable carrier, diluent or excipient.

In yet another aspect, the invention provides a method of inhibiting ordestroying a cell having a specific protease, such as an inflammatorycell or a cancer cell, comprising the steps of preparing a recombinantprotein of the invention having a heterologous linker sequence whichcontains a cleavage recognition site for the specific protease, andadministering the recombinant protein to the cells. In an embodiment,the inflammatory state is rheumatoid arthritis, atherosclerotic cells,Crohn's disease, or central nervous system disease. In anotherembodiment, the cancer is T-cell or B-cell lymphoproliferative disease,ovarian cancer, pancreatic cancer, head and neck cancer, squamous cellcarcinoma, gastrointestinal cancer, breast cancer, prostate cancer, nonsmall cell lung cancer.

The present invention also relates to a method of treating a cell havinga specific protease such as an inflammatory cell or a cancer cell,wherein the cells affected by the condition and which have a specificprotease, are treated by administering an effective amount of one ormore recombinant proteins of the invention to an animal in need thereof.

Still further, a process is provided for preparing a pharmaceutical fortreating a cell having a specific protease, such as an inflammatory cellor a cancer cell, wherein cells affected by condition have a specificprotease, the steps for preparing the pharmaceutical comprising thesteps of preparing a purified and isolated nucleic acid having anucleotide sequence encoding an A chain of a ricin-like toxin, a B chainof a ricin-like toxin and a heterologous linker amino acid sequence,linking the A and B chains, wherein the linker sequence contains acleavage recognition site for the specific protease; introducing thenucleic acid into a host cell; expressing the nucleic acid in the hostcell to obtain a recombinant protein comprising an A chain of aricin-like toxin, a B chain of a ricin-like toxin and a heterologouslinker amino acid sequence, linking the A and B chains wherein thelinker sequence contains the cleavage recognition site for the specificprotease; and suspending the protein in a pharmaceutically acceptablecarrier, diluent or excipient.

Other features and advantages of the present invention will becomeapparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples while indicating preferred embodiments of the invention aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

DESCRIPTION OF THE DRAWINGS

The invention will be better understood with reference to the drawingsin which:

FIG. 1A shows the nucleotide sequence of the MMP-9 linker region ofpAP301 (SEQ ID NOS:1-4);

FIG. 1B shows the nucleotide sequence of the pAP301 insert containingricin and the MMP-9 linker (SEQ ID NO:5);

FIG. 1C shows the amino acid sequence of the PAP301 linker and the wildtype ricin linker (SEQ ID NOS:6-7);

FIG. 2A shows the nucleotide sequence of the MMP-9 30 linker region ofpAP302 (SEQ ID NOS:8-11);

FIG. 2B shows the nucleotide sequence of the pAP302 insert containingricin and the MMP-9 linker (SEQ ID NO:12);

FIG. 2C shows the amino acid sequence of the PAP302 linker and the wildtype ricin linker (SEQ ID NOS:13-14);

FIG. 3A shows the nucleotide sequence of the MMP-9 linker region ofpAP303 (SEQ ID NOS:15-18);

FIG. 3B shows the nucleotide sequence of the pAP303 insert containingricin and the MMP-9 linker (SEQ ID NO:19);

FIG. 3C shows the amino acid sequence of the PAP303 linker and the wildtype ricin linker (SEQ ID NOS:20-21);

FIG. 4A shows the nucleotide sequence of the MMP-9 linker region ofpAP304 (SEQ ID NOS:22-25);

FIG. 4B shows the nucleotide sequence of the pAP304 insert containingricin and the MMP-9 linker (SEQ ID NO:26);

FIG. 4C shows the amino acid sequence of the PAP304 linker and the wildtype ricin linker (SEQ ID NOS:27-28);

FIG. 5A shows the nucleotide sequence of the MMP-9 linker region ofpAP305 (SEQ ID NOS:29-32);

FIG. 5B shows the nucleotide sequence of the pAP305 insert containingricin and the MMP-9 linker (SEQ ID NO:33);

FIG. 5C shows the amino acid sequence of the PAP305 linker and the wildtype ricin linker (SEQ ID NOS:34-35);

FIG. 6A shows the nucleotide sequence of the MMP-9 linker region ofpAP308 (SEQ ID NOS:36-39);

FIG. 6B shows the nucleotide sequence of the pAP308 insert containingricin and the MMP-9 linker (SEQ ID NO:40);

FIG. 6C shows the amino acid sequence of the pAP308 linker and the wildtype ricin linker (SEQ ID NOS:41-42);

FIG. 7A shows the nucleotide sequence of the MMP-9 linker region ofpAP309 (SEQ ID NOS:43-46);

FIG. 7B shows the nucleotide sequence of the pAP309 insert containingricin and the MMP-9 linker (SEQ ID NO:47);

FIG. 7C shows the amino acid sequence of the PAP309 linker and the wildtype ricin linker (SEQ ID NOS:48-49);

FIG. 8A shows the nucleotide sequence of the UPA linker region of pAP313(SEQ ID NOS:50-53);

FIG. 8B shows the nucleotide sequence of the pAP313 insert containingricin and the UPA linker (SEQ ID NO:54);

FIG. 8C shows the amino acid sequence of the PAP313 linker and the wildtype ricin linker (SEQ ID NOS:55-56);

FIG. 9A shows the nucleotide sequence of the UPA linker region of pAP314(SEQ ID NOS:57-60);

FIG. 9B shows the nucleotide sequence of the pAP314 insert containingricin and the UPA linker (SEQ ID NO:61);

FIG. 9C shows the amino acid sequence of the PAP314 linker and the wildtype ricin linker (SEQ ID NOS:62-63);

FIG. 10A shows the nucleotide sequence of the UPA linker region ofpAP315 (SEQ ID NOS:64-67);

FIG. 10B shows the nucleotide sequence of the pAP315 insert containingricin and the UPA linker (SEQ ID NO:68);

FIG. 10C shows the amino acid sequence of the PAP315 linker and the wildtype ricin linker (SEQ ID NOS:69-70);

FIG. 11A shows the nucleotide sequence of the MMP-9 linker region ofpAP316 (SEQ ID NOS:71-74);

FIG. 11B shows the nucleotide sequence of the pAP316 insert containingricin and the MMP-9 linker (SEQ ID NO:75);

FIG. 11C shows the amino acid sequence of the PAP316 linker and the wildtype ricin linker (SEQ ID NOS:76-77);

FIG. 12A shows the nucleotide sequence of the MMP-9 linker region ofpAP318 (SEQ ID NOS:78-81);

FIG. 12B shows the nucleotide sequence of the pAP318 insert containingricin and the MMP-9 linker (SEQ ID NO:82);

FIG. 12C shows the amino acid sequence of the PAP318 linker and the wildtype ricin linker (SEQ ID NOS:83-84);

FIG. 13A shows the nucleotide sequence of the UPA linker region ofpAP320 (SEQ ID NOS:85-88);

FIG. 13B shows the nucleotide sequence of the pAP320 insert containingricin and the UPA linker (SEQ ID NO:89);

FIG. 13C shows the amino acid sequence of the PAP320 linker and the wildtype ricin linker (SEQ ID NOS:90-91);

FIG. 14A shows the nucleotide sequence of the UPA linker region ofpAP321 (SEQ ID NOS:92-95);

FIG. 14B shows the nucleotide sequence of the pAP321 insert containingricin and the UPA linker (SEQ ID NO:96);

FIG. 14C shows the amino acid sequence of the PAP321 linker and the wildtype ricin linker (SEQ ID NOS:97-98);

FIG. 15A shows the nucleotide sequence of the UPA linker region ofpAP322 (SEQ ID NOS:99-102);

FIG. 15B shows the nucleotide sequence of the pAP322 insert containingricin and the UPA linker (SEQ ID NO:103);

FIG. 15C shows the amino acid sequence of the PAP322 linker and the wildtype ricin linker (SEQ ID NOS:104-105);

FIG. 16A shows the nucleotide sequence of the MMP-9 linker region ofpAP323 (SEQ ID NOS:106-109);

FIG. 16B shows the nucleotide sequence of the pAP323 insert containingricin and the MMP-9 linker (SEQ ID NO:110);

FIG. 16C shows the amino acid sequence of the PAP323 linker and the wildtype ricin linker (SEQ ID NOS:111-112);

FIG. 17A shows the nucleotide sequence of the MMP-9 linker region ofpAP324 (SEQ ID NOS:113-116);

FIG. 17B shows the nucleotide sequence of the pAP324 insert containingricin and the MMP-9 linker (SEQ ID NO:117);

FIG. 17C shows the amino acid sequence of the PAP324 linker and the wildtype ricin linker (SEQ ID NOS:118-119);

FIG. 18A shows the nucleotide sequence of the MMP-9 linker region ofpAP325 (SEQ ID NOS:120-123);

FIG. 18B shows the nucleotide sequence of the pAP325 insert containingricin and the MMP-9 linker (SEQ ID NO:124);

FIG. 18C shows the amino acid sequence of the PAP325 linker and the wildtype ricin linker (SEQ ID NOS:125-126);

FIG. 19 shows the cleavage products of an MMP-9 digestion of PAP323,PAP324 and PAP325;

FIG. 20 is a graph showing the treatment of human tumour A431 withPAP304;

FIG. 21 is a graph showing the treatment of human tumour A431 withPAP305; and

FIG. 22 is a graph showing a significant delay in tumor growth in themurine tumor model.

DETAILED DESCRIPTION OF THE INVENTION 1. Nucleic Acid Molecules of theInvention

As mentioned above, the present invention relates to isolated andpurified nucleic acid molecules encoding linker sequences. The presentinvention also relates to isolated and purified nucleic acid moleculesencoding a recombinant toxic protein comprising (a) a nucleotidesequence encoding an A chain of a ricin-like toxin, (b) a nucleotidesequence encoding a B chain of a ricin-like toxin and (c) a nucleotidesequence encoding a linker amino acid sequence of the invention, linkingthe A and B chains. The heterologous linker sequence contains a cleavagerecognition site for a specific protease.

The term “isolated and purified” as used herein refers to a nucleic acidsubstantially free of cellular material or culture medium when producedby recombinant DNA techniques, or chemical precursors, or otherchemicals when chemically synthesized. An “isolated and purified”nucleic acid is also substantially free of sequences which naturallyflank the nucleic acid (i.e. sequences located at the 5′ and 3′ ends ofthe nucleic acid) from which the nucleic acid is derived. The term“nucleic acid” is intended to include DNA and RNA and can be eitherdouble stranded or single stranded.

The term “linker sequence” as used herein refers to an internal aminoacid sequence within the protein encoded by a nucleic acid molecule ofthe invention which contains residues linking the A and B chain of aricin-like toxin so as to render the A chain incapable of exerting itstoxic effect, for example catalytically inhibiting translation of aneukaryotic ribosome. The linker sequences of the invention areheterologous to the A and B chain of a ricin-like toxin. By heterologousis meant that the linker sequence is not a sequence native to the A or Bchain of a ricin-like toxin or precursor thereof. However, preferably,the linker sequence may be of a similar length to the linker sequence ofa ricin-like toxin and should not interfere with the role of the B chainin cell binding and transport into the cytoplasm. When the linkersequence is cleaved the A chain becomes active or toxic.

The nucleic acid molecule of the invention encoding a recombinant toxicprotein is cloned by subjecting a preproricin cDNA clone tosite-directed mutagenesis in order to generate a series of variantsdiffering only in the sequence between the A and B chains (linkerregion). Oligonucleotides, corresponding to the extreme 5′ and 3′ endsof the preproricin gene are synthesized and used to PCR amplify thegene. Using the cDNA sequence for preproricin (Lamb et al., Eur. J.Biochem. 145:266-270 (1985)), several oligonucleotide primers aredesigned to flank the start and stop codons of the preproricin openreading frame.

The preproricin cDNA is amplified using the upstream primer Ricin-99 orRicin-109 and the downstream primer Ricin1729C with Vent DNA polymerase(New England Biolabs) using standard procedures (Sambrook et al.,Molecular Cloning: A Laboratory Manual, Second Edition, (Cold SpringHarbor Laboratory Press, 1989)). The purified PCR fragment encoding thepreproricin cDNA is, then ligated into an Eco RV-digested pBluescript IISK plasmid (Stratagene), and is used to transform competent XL1-Bluecells (Stratagene). The cloned PCR product containing the putativepreproricin gene is confirmed by DNA sequencing of the entire cDNAclone.

The preproricin cDNA clone is subjected to site directed mutagenesis; inorder to generate a series of variants differing only in the sequencebetween the A and B chains (linker region). The wild-type preproricinlinker region is replaced with the heterogenous linker sequences thatare cleaved by the various specific proteases.

The linker regions of the variants encode a cleavage recognitionsequence for a specific protease. The mutagenesis and cloning strategiesused to generate a specific protease-sensitive linker variant aresummarized in WO 98149311 to the present inventor. Briefly, the firststep involves a DNA amplification using a set of mutagenic primers incombination with the two flanking primers Ricin-109Eco and Ricin1729CPstI. Restriction digested PCR fragments are gel purified and thenligated with PVL1393 which has been digested with Eco RI and PstI.Ligation reactions are used to transform competent XLI-Blue cells(Stratagene). Recombinant clones are identified by restriction digestsof plasmid miniprep, DNA and the mutant linker sequences are confirmedby DNA sequencing.

The nucleotide sequences of the novel linker sequences of the inventionare as follows: the nucleic acid sequence of pAP301 is shown in FIG. 1A;the nucleic acid sequence of pAP302 is shown in FIG. 2A; the nucleicacid sequence of pAP303 is shown in FIG. 3A; the nucleic acid sequenceof pAP304 is shown in FIG. 4A; the nucleic acid sequence of pAP305 isshown in FIG. 5A; the nucleic acid sequence of pAP308 is shown in FIG.6A; the nucleic acid sequence of pAP309 is shown in FIG. 7A; the nucleicacid sequence of pAP313 is shown in FIG. 8A; the nucleic acid sequenceof pAP314 is shown in FIG. 9A; the nucleic acid sequence of pAP315 isshown in FIG. 10A; the nucleic acid sequence of pAP316 is shown in FIG.11A; the nucleic acid sequence of pAP318 is shown in FIG. 12A; thenucleic acid sequence of pAP320 is shown in FIG. 13A; the nucleic acidsequence of pAP321 is shown in FIG. 14A; the nucleic acid sequence ofpAP322 is shown in FIG. 15A; the nucleic acid sequence of pAP323 isshown in FIG. 16A; the nucleic acid sequence of pAP324 is shown in FIG.17A; and the nucleic acid sequence of pAP325 is shown in FIG. 18A.

The nucleic acid molecule encoding a recombinant protein of theinvention has sequences encoding an A chain of a ricin-like toxin, a Bchain of a ricin-like toxin and a heterologous linker sequencecontaining a cleavage recognition site for a specific protease asdescribed above. The nucleotide sequences encoding the recombinantproteins of the invention are shown in FIGS. 1B-18B. The nucleic acidmay be expressed to provide a recombinant protein having an A chain of aricin-like toxin, a B chain of a ricin-like toxin and a heterologouslinker sequence containing a cleavage recognition site for a specificprotease.

The nucleic acid molecule may comprise the A and/or B chain of ricin.The ricin gene has been cloned and sequenced, and the X-ray crystalstructures of the A and B chains are published (Rutenber, E., et al.Proteins 10:240-250 (1991); Weston et al., Mol. Biol. 244:410-422(1994); Lamb and Lord, Eur. J. Biochem. 14:265 (1985); Halling, K., etal., Nucleic Acids Res. 13:8019 (1985)). It will be appreciated that theinvention includes nucleic acid molecules encoding truncations of A andB chains of ricin-like proteins and analogs and homologs of A and Bchains of ricin-like proteins and truncations thereof (i.e., ricin-likeproteins), as described herein. It will further be appreciated thatvariant forms of the nucleic acid molecules of the invention which ariseby alternative splicing of an mRNA corresponding to a cDNA of theinvention are encompassed by the invention.

Another aspect of the invention provides a nucleotide sequence whichhybridizes under high stringency conditions to a nucleotide sequenceencoding the A and/or B chains of a ricin-like protein. Appropriatestringency conditions which promote DNA hybridization are known to thoseskilled in the art, or can be found in Current Protocols in MolecularBiology, John Wiley & Sons, N.Y. (1989), 6.3.1 6.3.6. For example, 6.0×sodium chloride/sodium citrate (SSC) at about 45° C., followed by a washof 2.0×SSC at 50° C. may be employed.

The stringency may be selected based on the conditions used in the washstep. By way of example, the salt concentration in the wash step can beselected from a high stringency of about 0.2×SSC at 50° C. In addition,the temperature in the wash step can be at high stringency conditions,at about 65° C.

The nucleic acid molecule may comprise the A and/or B chain of aricin-like toxin. Methods for cloning ricin-like toxins are known in theart and are described, for example, in E.P. 466,222. Sequences encodingricin or ricin-like A and B chains may be obtained by selectiveamplification of a coding region, using sets of degenerative primers orprobes for selectively amplifying the coding region in a genomic or cDNAlibrary. Appropriate primers may be selected from the nucleic acidsequence of A and B chains of ricin or ricin-like toxins. It is alsopossible to design synthetic oligonucleotide primers from the nucleotidesequences for use in PCR. Suitable primers may be selected from thesequences encoding regions of ricin-like proteins which are highlyconserved, as described for example in U.S. Pat. No. 5,101,025 and E.P.466,222.

A nucleic acid can be amplified from cDNA or genomic DNA using theseoligonucleotide primers and standard PCR amplification techniques. Thenucleic acid so amplified can be cloned into an appropriate vector andcharacterized by DNA sequence analysis. It will be appreciated that cDNAmay be prepared from mRNA, by isolating total cellular mRNA by a varietyof techniques, for example, by using the guanidinium-thiocyanateextraction procedure of Chirgwin et al. (Biochemistry 18, 5294-5299(1979)). cDNA is then synthesized from the mRNA using reversetranscriptase (for example, Moloney MLV reverse transcriptase availablefrom Gibco/BRL, Bethesda, Md., or AMV reverse transcriptase availablefrom Seikagaku America, Inc., St. Petersburg, Fla.). It will beappreciated that the methods described above may be used to obtain thecoding sequence from plants, bacteria or fungi, preferably plants, whichproduce known ricin-like proteins and also to screen for the presence ofgenes encoding as yet unknown ricin-like proteins.

A sequence containing a cleavage recognition site for a specificprotease may be selected based on the disease or condition which is tobe targeted by the recombinant protein. The cleavage recognition sitemay be selected from sequences known to encode a cleavage recognitionsite specific proteases of the disease or condition to be treated.Sequences encoding cleavage recognition sites may be identified bytesting the expression product of the sequence for susceptibility tocleavage by the respective protease. A polypeptide containing thesuspected cleavage recognition site may be incubated with a specificprotease and the amount of cleavage product determined (Dilannit, 1990,J. Biol. Chem. 285: 17345-17354 (1990)). The specific protease may beprepared by methods known in the art and used to test suspected cleavagerecognition sites.

The nucleic acid molecule of the invention may be prepared by sitedirected mutagenesis. For example, the cleavage site of a specificprotease may be prepared by site directed mutagenesis of the homologouslinker sequence of a proricin-like toxin. Procedures for cloningproricin-like genes, encoding a linker sequence are described in EP466,222. Site directed mutagenesis may be accomplished by DNAamplification of mutagenic primers in combination with flanking primers.

The nucleic acid molecule of the invention may also encode a fusionprotein. A sequence encoding a heterologous linker sequence containing acleavage recognition site for a specific protease may be cloned from acDNA or genomic library or chemically synthesized based on the knownsequence of such cleavage sites. The heterologous linker sequence maythen be fused in frame with the sequences encoding the A and B chains ofthe ricin-like toxin for expression as a fusion protein. It will beappreciated that a nucleic acid molecule encoding a fusion protein maycontain a sequence encoding an A chain and a B chain from the samericin-like toxin or the encoded A and B chains may be from differenttoxins. For example, the A chain may be derived from ricin and the Bchain may be derived from abrin. A protein may also be prepared bychemical conjugation of the A and B chains and linker sequence usingconventional coupling agents for covalent attachment.

An isolated and purified nucleic acid molecule of the invention which isRNA can be isolated by cloning a cDNA encoding an A and B chain and alinker into an appropriate vector which allows for transcription of thecDNA to produce an RNA molecule which encodes a protein of theinvention. For example, a cDNA can be cloned downstream of abacteriophage promoter, (e.g. a T7 promoter) in a vector, cDNA can betranscribed in vitro with T7 polymerase, and the resultant RNA can beisolated by standard techniques.

II. Novel Linkers and Recombinant Proteins of the Invention

As previously mentioned, the invention provides novel linker sequences.Preferably, the amino acid sequence of the linker is selected from: theamino acid sequence of PAP301 as shown in FIG. 1C; the amino acidsequence of PAP302 as shown in FIG. 2C; the amino acid sequence ofPAP303 as shown in FIG. 3C; the amino acid sequence of PAP304 as shownin FIG. 4C; the amino acid sequence of PAP305 as shown in FIG. 5C; theamino acid sequence of PAP308 as shown in FIG. 6C; the amino acidsequence of PAP309 as shown in FIG. 7C; the amino acid sequence ofPAP313 as shown in FIG. 8C; the amino acid sequence of PAP314 as shownin FIG. 9C; the amino acid sequence of PAP315 as shown in FIG. 10C; theamino acid sequence of PAP316 as shown in FIG. 11C; the amino acidsequence of PAP318 as shown in FIG. 12C; the amino acid sequence ofPAP320 as shown in FIG. 13C; the amino acid sequence of PAP321 as shownin FIG. 14C; the amino acid sequence of PAP322 as shown in FIG. 15C; theamino acid sequence of PAP323 as shown in FIG. 16C; the amino acidsequence of PAP324 as shown in FIG. 17C; and the amino acid sequence ofPAP325 as shown in FIG. 18C.

The present invention also provides recombinant proteins whichincorporate the A and B chains of a ricin-like toxin linked by aheterologous linker sequence containing a cleavage recognition site fora specific protease as described above. It is an advantage of therecombinant proteins of the invention that they are non-toxic until theA chain is liberated from the B chain by specific cleavage of the linkerby the target specific protease.

The recombinant protein may be used to specifically target for example,cancer cells in the absence of additional specific cell-bindingcomponents to target cancer cells. It is a further advantage that thespecific protease cleaves the heterologous linker intracellularlythereby releasing the toxic A chain directly into the cytoplasm of thetarget cell. As a result, said cells are specifically targeted andnormal cells are not directly exposed to the activated free A chain.

Ricin is a plant derived ribosome inhibiting protein which blocksprotein synthesis in eukaryotic cells. Ricin may be derived from theseeds of Ricinus communis (castor oil plant). The ricin toxin is aglycosylated heterodimer with A and B chain molecular masses of 30,625Da and 31,431 Da respectively. The A chain of ricin has an N-glycosidaseactivity and catalyzes the excision of a specific adenine residue fromthe 28S rRNA of eukaryotic ribosomes (Endo, Y; & Tsurugi, K. J. Biol.Chem. 262:8128 (1987)). The B chain of ricin, although not toxic initself, promotes the toxicity of the A chain by binding to galactoseresidues on the surface of eukaryotic cells and stimulatingreceptor-mediated endocytosis of the toxin molecule (Simmons et al.,Biol. Chem. 261:7912 (1986)).

All protein toxins are initially produced in an inactive, precursorform. Ricin is initially produced as a single polypeptide (preproricin)with a 35 amino acid N-terminal presequence and 12 amino acid linkerbetween the A and B chains. The pre-sequence is removed duringtranslocation of the ricin precursor into the endoplasmic reticulum(Lord, J. M., Eur. J. Biochem. 146:403-409 (1985) and Lord, J. M., Eur.J. Biochem. 146:411-416 (1985)). The proricin is then translocated intospecialized organelles called protein bodies where a plant proteasecleaves the protein at a linker region between the A and B chains (Lord,J. M. et al., FASAB journal 8:201-208 (1994)). The two chains, however,remain covalently attached by an interchain disulfide bond (cysteine 259in the A chain to cysteine 4 in the B chain) and mature disulfide linkedricin is stored in protein bodies inside plant cells. The A chain isinactive in the proricin (O'Hare, M., et al., FEBS Lett. 273:200-204(1990)) and it is inactive in the disulfide-linked mature ricin(Richardson, P. T. et al., FEBS Lett. 255:15-20 (1989)). The ribosomesof the castor bean plant are themselves susceptible to inactivation byricin A chain; however, as there is no cell surface galactose to permitB chain recognition the A chain cannot re-enter the cell.

Ricin-like proteins include, but are not limited to, bacterial, fungaland plant toxins which have A and B chains and inactivate ribosomes andinhibit protein synthesis. The A chain is an active polypeptide subunitwhich is responsible for the pharmacologic effect of the toxin. In mostcases the active component of the A chain is an enzyme. The B chain isresponsible for binding the toxin to the cell surface and is thought tofacilitate entry of the A chain into the cell cytoplasm. The A and Bchains in the mature toxins are linked by disulfide bonds. The toxinsmost similar in structure to ricin are plant toxins which have one Achain and one B chain. Examples of such toxins include abrin which maybe isolated from the seeds of Abrus precatorius, modeccin, volkensin andviscumin.

Ricin-like bacterial proteins include diphtheria toxin, which isproduced by Corynebacterium diphtheriae, Pseudomonas exotoxin andcholera toxin. It will be appreciated that the term ricin-like toxins isalso intended to include the A chain of those toxins which have only anA chain. The recombinant proteins of the invention could include the Achain of these toxins conjugated to, or expressed as, a recombinantprotein with the B chain of another toxin. Examples of plant toxinshaving only an A chain include trichosanthin, MMC and pokeweed antiviralproteins, dianthin 30, dianthin 32, crotin II, curcin 11 and wheat germinhibitor. Examples of fungal toxins having only an A chain includealpha-sarcin, restrictocin, mitogillin, enomycin, phenomycin. Examplesof bacterial toxins having only an A chain include cytotoxin fromShigella dysenteriae and related Shiga-like toxins. Recombinanttrichosanthin and the coding sequence thereof is disclosed in U.S. Pat.Nos. 5,101,025 and 5,128,460.

In addition to the entire A or B chains of a ricin-like toxin, it willbe appreciated that the recombinant protein of the invention may containonly that portion of the A chain which is necessary for exerting itscytotoxic effect. For example, the first 30 amino acids of the ricin Achain may be removed resulting in a truncated A chain which retainstoxic activity. The truncated ricin or ricin-like A chain may beprepared by expression of a truncated gene or by proteolyticdegradation, for example with Nagarase (Funmatsu et al., Jap. J. Med.Sci. Biol. 23:264-267 (1970)). Similarly, the recombinant protein of theinvention may contain only that portion of the B chain necessary forgalactose recognition, cell binding and transport into the cellcytoplasm. Truncated B chains are described for example in E.P. 145,111.The A and B chains may be glycosylated or non-glycosylated. GlycosylatedA and B chains may be obtained by expression in the appropriate hostcell capable of glycosylation. Non-glycosylated chains may be obtainedby expression in nonglycosylating host cells or by treatment to removeor destroy the carbohydrate moieties.

The proteins of the invention may be prepared using recombinant DNAmethods. Accordingly, the nucleic acid molecules of the presentinvention may be incorporated in a known manner into an appropriateexpression vector which ensures good expression of the protein. Possibleexpression vectors include but are not limited to cosmids, plasmids, ormodified viruses (e.g. replication defective retroviruses, adenovirusesand adeno-associated viruses), so long as the vector is compatible withthe host cell used. The expression vectors are “suitable fortransformation of a host cell”, which means that the expression vectorscontain a nucleic acid molecule of the invention and regulatorysequences selected on the basis of the host cells to be used forexpression, which is operatively linked to the nucleic acid molecule.Operatively linked is intended to mean that the nucleic acid is linkedto regulatory sequences in a manner which allows expression of thenucleic acid.

The invention therefore contemplates a recombinant expression vector ofthe invention containing a nucleic acid molecule of the invention, or afragment thereof, and the necessary regulatory sequences for thetranscription and translation of the inserted protein-sequence.

Suitable regulatory sequences may be derived from a variety of sources,including bacterial, fungal, viral, mammalian, or insect genes (Forexample, see the regulatory sequences described in Goeddel, GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990). Selection of appropriate regulatory sequences isdependent on the host cell chosen as discussed below, and may be readilyaccomplished by one of ordinary skill in the art. Examples of suchregulatory sequences include: a transcriptional promoter and enhancer orRNA polymerase binding sequence, a ribosomal binding sequence, includinga translation initiation signal. Additionally, depending on the hostcell chosen and the vector employed, other sequences, such as an originof replication, additional DNA restriction sites, enhancers, andsequences conferring inducibility of transcription may be incorporatedinto the expression vector. It will also be appreciated that thenecessary regulatory sequences may be supplied by the native A and Bchains and/or its flanking regions.

The recombinant expression vectors of the invention may also contain aselectable marker gene which facilitates the selection of host cellstransformed or transfected with a recombinant molecule of the invention.Examples of selectable marker genes are genes encoding a protein such asG418 and hygromycin which confer resistance to certain drugs,β-galactosidase, chloramphenicol acetyltransferase, firefly luciferase,or an immunoglobulin or portion thereof such as the Fc portion of animmunoglobulin preferably IgG. Transcription of the selectable markergene is monitored by changes in the concentration of the selectablemarker protein such as β-galactosidase, chloramphenicolacetyltransferase, or firefly luciferase. If the selectable marker geneencodes a protein conferring antibiotic resistance such as neomycinresistance transformant cells can be selected with G418. Cells that haveincorporated the selectable marker gene will survive, while the othercells die. This makes it possible to visualize and assay for expressionof recombinant expression vectors of the invention and in particular todetermine the effect of a mutation on expression and phenotype. It willbe appreciated that selectable markers can be introduced on a separatevector from the nucleic acid of interest.

The recombinant expression vectors may also contain genes which encode afusion moiety which provides increased expression of the recombinantprotein; increased solubility of the recombinant protein; and aid in thepurification of the target recombinant protein by acting as a ligand inaffinity purification. For example, a proteolytic cleavage site may beadded to the target recombinant protein to allow separation of therecombinant protein from the fusion moiety subsequent to purification ofthe fusion protein. Typical fusion expression vectors include pGEX(Amrad Corp., Melbourne, Australia), pMal (New England Biolabs, Beverly,Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathioneS-transferase (GST), maltose E binding protein, or protein A,respectively, to the recombinant protein.

Recombinant expression vectors can be introduced into host cells toproduce a transformed host cell. The term “transformed host cell” isintended to include prokaryotic and eukaryotic cells which have beentransformed or transfected with a recombinant expression vector of theinvention. The terms “transformed with”, “transfected with”,“transformation” and “transfection” are intended to encompassintroduction of nucleic acid (e.g. a vector) into a cell by one of manypossible techniques known in the art. Prokaryotic cells can betransformed with nucleic acid by, for example, electroporation orcalcium-chloride mediated transformation. Nucleic acid can be introducedinto mammalian cells via conventional techniques such as calciumphosphate or calcium chloride co-precipitation, DEAE-dextran mediatedtransfection, lipofectin, electroporation or microinjection. Suitablemethods for transforming and transfecting host cells can be found inSambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition,Cold Spring Harbor Laboratory press (1989)), and other laboratorytextbooks.

Suitable host cells include a wide variety of prokaryotic and eukaryotichost cells. For example, the proteins of the invention may be expressedin bacterial cells such as E. coli, insect cells (using baculovirus),yeast cells or mammalian cells. Other suitable host cells can be foundin Goeddel, Gene Expression Technology: Methods in Enzymology 185,Academic Press, San Diego, Calif. (1991).

More particularly, bacterial host cells suitable for carrying out thepresent invention include E. coli, B. subtilis, Salmonella typhimurium,and various species within the genus Pseudomonas, Streptomyces, andStaphylococcus, as well as many other bacterial species well known toone of ordinary skill in the art. Suitable bacterial expression vectorspreferably comprise a promoter which functions in the host cell, one ormore selectable phenotypic markers, and a bacterial origin ofreplication. Representative promoters include the β-lactamase(penicillinase) and lactose promoter system (see Chang et al., Nature275:615 (1978)), the trp promoter (Nichols and Yanofsky, Meth inEnzymology 101:155, (1983) and the tac promoter (Russell et al., Gene20:231, (1982)). Representative selectable markers include variousantibiotic resistance markers such as the kanamycin or ampicillinresistance genes. Suitable expression vectors include but are notlimited to bacteriophages such as lambda derivatives or plasmids such aspBR322 (Bolivar et al., Gene 2:9 S, (1977)), the pUC plasmids pUC18,pUC19, pUC118, pUC119 (see Messing, Meth in Enzymology 101:20-77, 1983and Vieira and Messing, Gene 19:259-268 (1982)), and pNH8A, pNH16a,pNH18a, and Bluescript M13 (Stratagene, La Jolla, Calif.).

Typical fusion expression vectors which may be used are discussed above,e.g. pGEX (Amrad Corp., Melbourne, Australia), pMAL (New EnglandBiolabs, Beverly, Mass.) and pRITS (Pharmacia, Piscataway, N.J.).Examples of inducible non-fusion expression vectors include pTrc (Arnannet al., Gene 69:301-315 (1988)) and pET 11d (Studier et al., GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif., 60-89 (1990)).

Yeast and fungi host cells suitable for carrying out the presentinvention include, but are not limited to Saccharomyces cerevisae, thegenera Pichia or Kluyveromyces and various species of the genusAspergillus. Examples of vectors for expression in yeast S. cerivisaeinclude pYepSec1 (Baldari. et al., Embo J. 6:229-234 (1987)), pMFa(Kurjan and Herskowitz, Cell 30:933-943 (1982)), pJRY88 (Schultz et al.,Gene 54:113-123 (1987)), and pYES2 (Invitrogen Corporation, San Diego,Calif.). Protocols for the transformation of yeast and fungi are wellknown to those of ordinary skill in the art (see Hinnen et al., Proc.Natl. Acad. Sci. USA 75:1929 (1978); Itoh et al., J. Bacteriology153:163 (1983), and Cullen et al. (Biol Technology 5:369 (1987)).

Mammalian cells suitable for carrying out the present invention include,among others: COS (e.g., ATCC No. CRL 1650 or 1651), BHK (e.g. ATCC No.CRL 6281), CHO (ATCC No. CCL 61), HeLa (e.g., ATCC No. CCL 2), 293 (ATCCNo. 1573) and NS-1 cells. Suitable expression vectors for directingexpression in mammalian cells generally include a promoter (e.g.,derived from viral material such as polyoma, Adenovirus 2,cytomegalovirus and Simian Virus 40), as well as other transcriptionaland translational control sequences. Examples of mammalian expressionvectors include pCDM8 (Seed, B., Nature 329:840 (1987)) and pMT2PC(Kaufman et al., EMBO J. 6:187-195 (1987)).

Given the teachings provided herein, promoters, terminators, and methodsfor introducing expression vectors of an appropriate type into plant,avian, and insect cells may also be readily accomplished. For example,within one embodiment, the proteins of the invention may be expressedfrom plant cells (see Sinkar et al., J. Biosci (Bangalore) 11:47-58(1987), which reviews the use of Agrobacterium rhizogenes vectors; seealso Zambryski et al., Genetic Engineering, Principles and Methods,Hollaender and Setlow (eds.), Vol. VI, pp. 253-278, Plenum Press, NewYork (1984), which describes the use of expression vectors for plantcells, including, among others, PAPS2022, PAPS2023, and PAPS2034)

Insect cells suitable for carrying out the present invention includecells and cell lines from Bombyx, Trichoplusia or Spodotera species.Baculovirus vectors available for expression of proteins in culturedinsect cells (SF 9 cells) include the pAc series (Smith et al., Mol.Cell. Biol. 3:2156-2165 (1983)) and the pVL series (Lucklow, V. A., andSummers, M. D., Virology 170:31-39 (1989)). Some baculovirus-insect cellexpression systems suitable for expression of the recombinant proteinsof the invention are described in PCT/US/02442.

Alternatively, the proteins of the invention may also be expressed innon-human transgenic animals such as, rats, rabbits, sheep and pigs(Hammer et al. Nature 315:680-683 (1985); Palmiter et al. Science222:809-814 (1983); Brinster et al. Proc. Natl. Acad. Sci. USA82:4438-4442 (1985); Palmiter and Brinster Cell 41:343-345 (1985) andU.S. Pat. No. 4,736,866).

The proteins of the invention may also be prepared by chemical synthesisusing techniques well known in the chemistry of proteins such as solidphase synthesis (Merrifield, J. Am. Chem. Assoc. 85:2149-2154 (1964)) orsynthesis in homogenous solution (Houbenweyl, Methods of OrganicChemistry, ed. E. Wansch, Vol. 15 I and II, Thieme, Stuttgart (1987)).

The present invention also provides proteins comprising an A chain of aricin-like toxin, a B chain of a ricin-like toxin and a heterologouslinker amino acid sequence linking the A and B chains, wherein thelinker sequence contains a cleavage recognition site for a specificprotease. Such a protein could be prepared other than by recombinantmeans, for example by chemical synthesis or by conjugation of A and Bchains and a linker sequence isolated and purified from their naturalplant, fungal or bacterial source. Such A and B chains could be preparedhaving the glycosylation pattern of the native ricin-like toxin.

N-terminal or C-terminal fusion proteins comprising the protein of theinvention conjugated with other molecules, such as proteins may beprepared by fusing, through recombinant techniques. The resultant fusionproteins contain a protein of the invention fused to the selectedprotein or marker protein as described herein. The recombinant proteinof the invention may also be conjugated to other proteins by knowntechniques. For example, the proteins may be coupled usingheterobifunctional thiol-containing linkers as described in WO 90/10457,N-succinimidyl-3-(2-pyridyldithio-proprionate) or N-succinimidyl-5thioacetate. Examples of proteins which may be used to prepare fusionproteins or conjugates include cell binding proteins such asimmunoglobulins, hormones, growth factors, lectins, insulin, low densitylipoprotein, glucagon, endorphins, transferrin, bombesin,asialoglycoprotein glutathione-S-transferase (GST), hemagglutinin (HA),and truncated myc.

III. Utility of the Nucleic Acid Molecules and Proteins of the Invention(a) Therapeutic Methods

As mentioned above, matrix metalloproteinases (MMPs or matrixins) arezinc-dependent proteinases and the expression of MMP genes is reportedto be activated in inflammatory disorders (e.g. rheumatoid arthritis)and malignancy. In addition, there are reports of increased activationand expression of urokinase type plasminogen activator in inflammatorydisorders such a rheumatoid arthritis (Slot, O., et al. 1999),osteoarthritis (Pap, G. et al., 2000), atherosclerotic cells(Falkenberg, M., et al., 1998) Crohn's disease (Desreumaux P, et al.1999), central nervous system disease (Cuzner and Opdenakker, 1999) aswell as in malignancy. Accordingly, the recombinant proteins of theinvention may be used to specifically inhibit or destroy cells thatcontain a specific protease that can cleave the linker sequence of therecombinant protein. More particularly, the recombinant proteins of theinvention may be used to specifically inhibit or destroy cancer cellsthat contain a protease that can cleave the linker sequence of therecombinant protein.

It is an advantage of the recombinant proteins of the invention thatthey have specificity for cells that contain a specific protease,including those of inflammatory disorders and cancer cells, without theneed for a cell binding component. The ricin-like B chain of therecombinant proteins recognize galactose moieties on the cell surfaceand ensure that the protein is taken up by, for example, a cancer celland released into the cytoplasm. When the protein is internalized into anormal cell, cleavage of the heterologous linker would not occur in theabsence of the specific protease, and the A chain will remain inactivebound to the B chain. Conversely, when the protein is internalized intoa cell having a specific protease, the specific protease will cleave thecleavage recognition site in the linker thereby releasing the toxic Achain.

Accordingly, the present invention provides a method of inhibiting ordestroying cells having a specific protease, for examples inflammatorycells or cancer cells, comprising contacting such cells with aneffective amount of a recombinant protein or a nucleic acid moleculeencoding a recombinant protein of the invention. The present inventionalso provides a method of treating a cell having a specific protease,comprising administering an effective amount of a recombinant protein ora nucleic acid molecule encoding a recombinant protein of the inventionto an animal in need thereof.

The term “effective amount” as used herein means an amount effective, atdosages and for periods of time necessary to achieve the desired result.

The term “animal” as used herein means any member of the animal kingdomincluding all mammals, birds, fish, reptiles and amphibians. Preferably,the animal to be treated is a mammal, more preferably a human.

The specificity of a recombinant protein of the invention may be testedby treating the protein with the specific protease which is thought tobe specific for the cleavage recognition site of the linker and assayingfor cleavage products. For example, specific proteases may be isolatedfrom cancer cells, or they may be prepared recombinantly, for examplefollowing the procedures in Darket et al. (J. Biol. Chem. 254:2307-2312(1988)). The cleavage products may be identified for example based onsize, antigenicity or activity. The toxicity of the recombinant proteinmay be investigated by subjecting the cleavage products to an in vitrotranslation assay in cell lysates, for example using Brome Mosaic VirusmRNA as a template. Toxicity of the cleavage products may be determinedusing a ribosomal inactivation assay (Westby et al., Bioconjugate Chem.3:377-382 (1992)). The effect of the cleavage products on proteinsynthesis may be measured in standardized assays of in vitro translationutilizing partially defined cell free systems composed for example of areticulocyte lysate preparation as a source of ribosomes and variousessential cofactors, such as mRNA template and amino acids. Use ofradiolabelled amino acids in the mixture allows quantitation ofincorporation of free amino acid precursors into trichloroacetic acidprecipitable proteins. Rabbit reticulocyte lysates may be convenientlyused (O'Hare, FEBS Lett. 273:200-204 (1990)).

The ability of the recombinant proteins of the invention to selectivelyinhibit or destroy cells having specific proteases may be readily testedin vitro using cell lines having the specific protease, such as cancercell lines. The selective inhibitory effect of the recombinant proteinsof the invention may be determined, for example, by demonstrating theselective inhibition of cellular proliferation in cancer cells orinfected cells.

Toxicity may also be measured based on cell viability, for example, theviability of cancer and normal cell cultures exposed to the recombinantprotein may be compared. Cell viability may be assessed by knowntechniques, such as trypan blue exclusion assays.

In another example, a number of models may be used to test thecytotoxicity of recombinant proteins having a heterologous linkersequence containing a cleavage recognition site for a cancer associatedmatrix metalloprotease. Thompson, E. W. et al. (Breast Cancer Res.Treatment 31:357-370 (1994)) has described a model for the determinationof invasiveness of human breast cancer cells in vitro by measuringtumour cell-mediated proteolysis of extracellular matrix and tumour cellinvasion of reconstituted basement membrane (collagen, laminin,fibronectin, Matrigel or gelatin). Other applicable cancer cell modelsinclude cultured ovarian adenocarcinoma cells (Young, T. N. et al.Gynecol. Oncol. 62:89-99 (1996); Moore, D. H. et al. Gynecol. Oncol.65:78-82 (1997)), human follicular thyroid cancer cells (Demeure, M. J.et al., World J. Surg. 16:770-776 (1992)), human melanoma (A-2058) andfibrosarcoma (HT-1080) cell lines (Mackay, A. R. et al. Lab. Invest.70:781 783 (1994)), and lung squamous (HS-24) and adenocarcinoma (SB-3)cell lines (Spiess, E. et al. J. Histochem. Cytochem. 42:917-929(1994)). An in vivo test system involving the implantation of tumoursand measurement of tumour growth and metastasis in athymic nude mice hasalso been described (Thompson, E. W. et al., Breast Cancer Res.Treatment 31:357-370 (1994); Shi, Y. E. et al., Cancer Res. 53:1409-1415(1993)).

Although the primary specificity of the proteins of the invention forcells having a specific protease is mediated by the specific cleavage ofthe cleavage recognition site of the linker, it will be appreciated thatspecific cell binding components may optionally be conjugated to theproteins of the invention. Such cell binding components may be expressedas fusion proteins with the proteins of the invention or the cellbinding component may be physically or chemically coupled to the proteincomponent. Examples of suitable cell binding components includeantibodies to cancer proteins.

Antibodies having specificity for a cell surface protein may be preparedby conventional methods. A mammal, (e.g. a mouse, hamster, or rabbit)can be immunized with an immunogenic form of the peptide which elicitsan antibody response in the mammal Techniques for conferringimmunogenicity on a peptide include conjugation to carriers or othertechniques well known in the art. For example, the peptide can beadministered in the presence of adjuvant. The progress of immunizationcan be monitored by detection of antibody titers in plasma or serum.Standard ELISA or other immunoassay procedures can be used with theimmunogen as antigen to assess the levels of antibodies. Followingimmunization, antisera can be obtained and, if desired, polyclonalantibodies isolated from the sera.

To produce monoclonal antibodies, antibody producing cells (lymphocytes)can be harvested from an immunized animal and fused with myeloma cellsby standard somatic cell fusion procedures thus immortalizing thesecells and yielding hybridoma cells. Such techniques are well known inthe art, (e.g. the hybridoma technique originally developed by Kohlerand Milstein (Nature 256:495-497 (1975)) as well as other techniquessuch as the human B-cell hybridoma technique (Kozbor et al., Immunol.Today 4:72 (1983)), the EBV-hybridoma technique to produce humanmonoclonal antibodies (Cole et al., Methods Enzymol, 121:140-67 (1986)),and screening of combinatorial antibody libraries (Huse et al., Science246:1275 (1989)). Hybridoma cells can be screened immunochemically forproduction of antibodies specifically reactive with the peptide and themonoclonal antibodies can be isolated.

The term “antibody” as used herein is intended to include fragmentsthereof which also specifically react with a cell surface component.Antibodies can be fragmented using conventional techniques and thefragments screened for utility in the same manner as described above.For example, F(ab′)₂ fragments can be generated by treating antibodywith pepsin. The resulting F(ab′)₂ fragment can be treated to reducedisulfide bridges to produce Fab fragments.

Chimeric antibody derivatives, i.e., antibody molecules that combine anon-human animal variable region and a human constant region are alsocontemplated within the scope of the invention. Chimeric antibodymolecules can include, for example, the antigen binding domain from anantibody of a mouse, rat, or other species, with human constant regions.Conventional methods may be used to make chimeric antibodies containingthe immunoglobulin variable region which recognizes a cell surfaceantigen (See, for example, Morrison et al., Proc. Natl. Acad. Sci.U.S.A. 81:6851 (1985); Takeda et al., Nature 314:452 (1985), Cabilly etal., U.S. Pat. No. 4,816,567; Boss et al., U.S. Pat. No. 4,816,397;Tanaguchi et al., E.P. Patent No. 171,496; European Patent No. 173,494;United Kingdom Patent No. GB 2177096B). It is expected that chimericantibodies would be less immunogenic in a human subject than thecorresponding non-chimeric antibody.

Monoclonal or chimeric antibodies specifically reactive against cellsurface components can be further humanized by producing human constantregion chimeras, in which parts of the variable regions, particularlythe conserved framework regions of the antigen-binding domain, are ofhuman origin and only the hypervariable regions are of non-human origin.Such immunoglobulin molecules may be made by techniques known in theart, (e.g. Teng et al., Proc. Natl. Acad. Sci. U.S.A., 80:7308-7312(1983); Kozbor et al., Immunology Today 4:7279 (1983); Olsson et al.,Meth. Enzymol., 92:3-16 (1982), and PCT Publication WO92/06193 or EP239,400). Humanized antibodies can also be commercially produced(Scotgen Limited, 2 Holly Road, Twickenham, Middlesex, Great Britain.)

Specific antibodies, or antibody fragments, reactive against cellsurface components may also be generated by screening expressionlibraries encoding immunoglobulin genes, or portions thereof, expressedin bacteria with cell surface components. For example, complete Fabfragments, VH regions and FV regions can be expressed in bacteria usingphage expression libraries (See for example Ward et al., Nature341:544-546 (1989); Huse et al., Science 246:1275-1281 (1989); andMcCafferty et al., Nature 348:552-554 (1990)). Alternatively, a SCID-humouse, for example the model developed by Genpharm, can be used toproduce antibodies, or fragments thereof.

(b) Pharmaceutical Compositions

The proteins and nucleic acids of the invention may be formulated intopharmaceutical compositions for administration to subjects in abiologically compatible form suitable for administration in vivo. By“biologically compatible form suitable for administration in vivo” ismeant a form of the substance to be administered in which any toxiceffects are outweighed by the therapeutic effects. The substances may beadministered to living organisms including humans, and animals.Administration of a therapeutically active amount of the pharmaceuticalcompositions of the present invention is defined as an amount effective,at dosages and for periods of time necessary to achieve the desiredresult. For example, a therapeutically active amount of a substance mayvary according to factors such as the disease state, age, sex, andweight of the individual, and the ability of antibody to elicit adesired response in the individual. Dosage regime may be adjusted toprovide the optimum therapeutic response. For example, several divideddoses may be administered daily or the dose may be proportionallyreduced as indicated by the exigencies of the therapeutic situation.

Accordingly, the present invention provides a pharmaceutical compositionfor treating cells having a specific protease comprising a recombinantprotein or a nucleic acid encoding a recombinant protein of theinvention and a pharmaceutically acceptable carrier, diluent orexcipient.

The active substance may be administered in a convenient manner such asby injection (subcutaneous, intravenous, intramuscular, etc.), oraladministration, inhalation, transdermal administration (such as topicalcream or ointment, etc.), or suppository applications. Depending on theroute of administration, the active substance may be coated in amaterial to protect the compound from the action of enzymes, acids andother natural conditions which may inactivate the compound.

The compositions described herein can be prepared by per se knownmethods for the preparation of pharmaceutically acceptable compositionswhich can be administered to subjects, such that an effective quantityof the active substance is combined in a mixture with a pharmaceuticallyacceptable vehicle. Suitable vehicles are described, for example, inRemington's Pharmaceutical Sciences (Remington's PharmaceuticalSciences, Mack Publishing Company, Easton, Pa., USA 1985). On thisbasis, the compositions include, albeit not exclusively, solutions ofthe substances in association with one or more pharmaceuticallyacceptable vehicles or diluents, and contained in buffered solutionswith a suitable pH and iso-osmotic with the physiological fluids.

The pharmaceutical compositions may be used in methods for treatinganimals, including mammals, preferably humans, with cancer. It isanticipated that the compositions will be particularly useful fortreating patients with B-cell lymphoproliferative disease and melanoma.The dosage and type of recombinant protein to be administered willdepend on a variety of factors which may be readily monitored in humansubjects. Such factors include the etiology and severity (grade andstage) of the neoplasia.

The following non-limiting examples are illustrative of the presentinvention:

EXAMPLES Example 1 Cloning and Expression of Proricin Variants Activatedby Disease Specific Proteases Isolation of Total RNA

The preproricin gene was cloned from new foliage of the castor beanplant. Total messenger RNA was isolated according to establishedprocedures (Sambrook et al., Molecular Cloning: A Lab Manual (ColdSpring Harbour Press, Cold Spring Harbour, (1989)) and cDNA generatedusing reverse transcriptase.

cDNA Synthesis

Oligonucleotides, corresponding to the extreme 5′ and 3′ ends of thepreproricin gene were synthesized and used to PCR amplify the gene.Using the cDNA sequence for preproricin (Lamb et al., Eur. J. Biochem.,145:266-270, 1985), several oligonucleotide primers were designed toflank the start and stop codons of the preproricin open reading frame.The oligonucleotides were synthesized using an Applied Biosystems Model392 DNA/RNA Synthesizer. First strand cDNA synthesis was primed usingthe oligonucleotide Ricin1729C. Three micrograms of total RNA was usedas a template for oligo Ricin1729C (5′-ATAACTTGCTGCTCCTTTCA-3′ (SEQ IDNO:127)) primed synthesis of cDNA using Superscript II ReverseTranscriptase (BRL) following the manufacturer's protocol.

DNA Amplification and Cloning

The first strand cDNA synthesis reaction was used as template for DNAamplification by the polymerase chain reaction (PCR). The preproricincDNA was amplified using the upstream primer Ricin-99(5′-CCGGGAGGAAATACTATTGTAAT-3′ (SEQ ID NO:128)) and the downstreamprimer Ricin1729C with Vent DNA polymerase (New England Biolabs) usingstandard procedures (Sambrook et al., Molecular Cloning: A LaboratoryManual, Second Edition, (Cold Spring Harbor Laboratory Press, 1989)).Amplification was carried out in a Biometra thermal cycler(TRIO-Thermalcycler) using the following cycling parameters:denaturation 95° C. for 1 min., annealing 52° C. for 1 min., andextension 72° C. for 2 min., (33 cycles), followed by a final extensioncycle at 72° C. for 10 min. The 1846 by amplified product wasfractionated on an agarose gel (Sambrook et al., Molecular Cloning: ALaboratory Manual, Second Edition, (Cold Spring Harbor Laboratory Press,1989), and the DNA purified from the gel slice using Qiaex resin(Qiagen) following the manufacturer's protocol. The purified PCRfragment encoding the preproricin cDNA was then ligated (Sambrook etal., Molecular Cloning: A Laboratory Manual, Second Edition, (ColdSpring Harbor Laboratory Press, 1989)) into an Eco RV digestedpBluescript II SK plasmid (Stratagene), and used to transform competentXL1-Blue cells (Stratagene). Positive clones were confirmed byrestriction digestion of purified plasmid DNA. Plasmid DNA was extractedusing a Qiaprep Spin Plasmid Miniprep Kit (Qiagen).

DNA Sequencing

The cloned PCR product containing the putative preproricin gene (pAP144)was confirmed by DNA sequencing of the entire cDNA clone. Sequencing wasperformed using an Applied Biosystems 373A Automated DNA Sequencer, andconfirmed by double-stranded dideoxy sequencing by the Sanger methodusing the Sequenase kit (USB) (see WO 98/49311).

Production and Cloning of Linker Variants

pAP144 cut with EcoR1 was used as target for PCR pairs employing theRicin109-Eco oligonucleotide (Ricin-109Eco primer:5-GGAGGAATCCGGAGATGAAACCGGGAGGAAATACTATTGTAAT-3 (SEQ ID NO:129)) and amutagenic primer for the 5′ half of the linker as well as theRicin1729PstI primer (Ricin 1729-PstI:5GTAGGCGCTGCAGATAACTTGCTGTCCTTTCAG-3 (SEQ ID NO:130)) and a mutagenicprimer for the 3′ half of the linker. The cycling conditions used forthe PCRs were 98 degrees C. for 2 min.; 98° C. 1 min., 52° C. 1 min.,72° C. 1 min. 15 sec. (30 cycles); 72 degrees C. 10 min.; 4 degrees C.soak. The PCR products were then digested by EcoRI and PstIrespectively, electrophoresed on an agarose gel, and the bands purifiedby via glass wool spin columns. Triple ligations comprising the PCRproduct pairs (corresponding halves of the new linker) and pVL1393vector digested with EcoR1 and PstI were carried out. Recombinant cloneswere identified by restriction digests of plasmid miniprep DNA and thealtered linkers confirmed by DNA sequencing. Note that all alteredlinker variants were cloned directly into the pVL1393 vector.

Isolation of Recombinant Baculoviruses

Insect cells S. frugiperda (Sf9), and Trichoplusia ni (Tn368 andBTI-TN-581-4 (High Five)) were maintained on EX-CELL 405 medium JRHBiosciences) supplemented with 10% total calf serum (Summers et al., AManual of Methods of Baculovirus Vectors and Insect Cell CultureProcedures, (Texas Agricultural Experiment Station, 1987)). Twomicrograms of recombinant pVL1393 DNA was co-transfected with 0.5microgram of BaculoGold AcNPV DNA (Pharmingen) into 2×10⁶ Tn368 insectcells following the manufacturer's protocol (Gruenwald et al.,Baculovirus Expression Vector System: Procedures and Methods Manual, 2ndEdition, (San Diego, Calif., 1993)). On day 5 post-transfection, mediawere centrifuged and the supernatants tested in limiting dilution assayswith Tn368 cells (Summers et al., A Manual of Methods of BaculovirusVectors and Insect Cell Culture Procedures, (Texas AgriculturalExperiment Station, 1987)). Recombinant viruses in the supernatants werethen amplified by infecting Tn368 cells at a multiplicity of infection(moi) of 0.1, followed by collection of day 3 to 5 supernatants. A totalof three rounds of amplification were performed for each recombinantfollowing established procedures (Summers et al., A Manual of Methods ofBaculovirus Vectors and Insect Cell Culture Procedures, (TexasAgricultural Experiment Station, 1987 and Gruenwald et al., BaculovirusExpression Vector System: Procedures and Methods Manual, 2nd Edition,(San Diego, Calif., 1993)).

Expression of Mutant Proricin

Recombinant baculoviruses were used to infect 1×10⁷ Tn368 or Sf9 cellsat an moi of 9 in EX-CELL 405 media (JRH Biosciences) with 25 mMa-lactose in spinner flasks. Media supernatants containing mutantproricins were collected 3 or 4 days post-infection.

Example 2 Harvesting and Affinity Column Purification of Pro-RicinVariants

Protein samples were harvested three days post infection. The cells wereremoved by centrifuging the media at 8288 g for ten minutes using a GS3(Sorvall) centrifuge rotor. The supernatant was further clarified bycentrifuging at 25400 g using a SLA-1500 rotor (Sorvall) for 45 minutes.Protease inhibitor phenylmethylsulfonyl fluoride (Sigma) was slowlyadded to a final concentration of 1 mM. The samples were furtherprepared by adding a-lactose to a concentration of 20 mM (not includingthe previous lactose contained in the expression medium). The sampleswere concentrated to 700 mL using a Prep/Scale-TFF Cartridge (2.5 ft,10K regenerated cellulose (Millipore)) and a Masterflex pump. Thesamples were then dialysed for 2 days in 1× Column Buffer (50 mM Tris,100 mM NaCl, 0.02% NaN3, pH 7.5) using dialysis tubing (10 K MWCO, 32 mmflat width (Spectra/Por)). Subsequently, the samples were clarified bycentrifuging at 25400 g using a SLA-1500 rotor (Sorvall) for 45 minutes.

Following centrifugation, the samples were degassed and applied at 4degrees C. to a XK26/20 (Pharmacia) column (attached to a Pharmaciaperistaltic pump, Pharmacia Single-path Monitor UV-1 Control and OpticalUnits, and Bromma LKB 2210 2-Channel Recorder) containing 20 mLa-Lactose Agarose Resin (Sigma). The column was washed for 3 hours with1× Column buffer. Elution of proricin variant was performed by elutingwith buffer (1× Column buffer (0.1% NaN₃), 100 mM Lactose) until thebaseline was again restored. The samples were concentrated using anAmicon 8050 concentrator (Amicon) with a YM10 76 mm membrane, utilizingargon gas to pressurize the chamber. The samples were furtherconcentrated in Centricon 10 (Millipore) concentrators according tomanufacturer's specifications.

Purification of Variant PAP-Protein by Gel Filtration Chromatography

In order to purify variant from processed material produced duringfermentation, the protein was applied to a SUPERDEX 75 (16/60) columnand SUPERDEX 200 (16/60) column (Pharmacia) connected in seriesequilibrated with 100 mM Tris, 200 mM NaCl, pH 7.5 containing 100 mMlactose and 1.0% β-mercaptoethanol (βME). The flow rate of the columnwas 0.15 mL/min and fractions were collected every 25 minutes. The UV(280 nm) trace was used to determine the approximate location of thepurified PAP-protein and thus determine the samples for Westernanalysis.

Western Analysis of Column Fractions

Fractions eluted from the SUPERDEX columns (Pharmacia) were analyzed forpurity using standard Western blotting techniques. An aliquot of 10 μLfrom each fraction was boiled in 1× sample buffer (62.6 mM Tris-Cl, pH6.8, 4.4% 13ME, 2% sodium dodecyl sulfate (SDS), 5% glycerol (all fromSigma) and 0.002% bromophenol blue (Biorad)) for five minutes. Denaturedsamples were loaded on 12% Tris-Glycine Gels (Biorad) along with 50 ngof RCA₆₀ (Sigma) and 5 μL of kaleidoscope prestained standards (Biorad).Electrophoresis was carried out for ninety minutes at 100V in 25 mMTris-Cl, pH 8.3, 0.1% SDS, and 192 mM glycine using the BioRad MiniProtean II cells (Biorad).

Following electrophoresis gels were equilibrated in transfer buffer (48mM Tris, 39 mM glycine, 0.0375% SDS, and 20% Methanol) for a fewminutes. PVDF Biorad membrane was presoaked for one minute in 100%methanol, rinsed in ddH₂O and two minutes in transfer buffer. Whatmanpaper was soaked briefly in transfer buffer. Five pieces of Whatmanpaper, membrane, gel, and another five pieces of Whatman paper werearranged on the bottom cathode (anode) of the Pharmacia Novablottransfer apparatus (Pharmacia). Transfer was for one hour at constantcurrent (2 mA/cm²).

Transfer was confirmed by checking for the appearance of the prestainedstandards on the membrane. Non-specific sites on the membrane wereblocked by incubating the blot for thirty minutes in 1× PhosphateBuffered Saline (1×PBS; 137 mM NaCl, 2.7 mM KCl, 8 mM Na₂HPO₄,1.5 mMKH₂PO₄, pH 7.4) with 5% skim milk powder (Carnation). Primary antibodyrabbit anti-ricin, (Sigma) was diluted 1:3000 in 1×PBS containing 0.1%Tween 20 (Sigma) and 2.5% skim milk and incubated with blot for fortyfive minutes on a orbital shaker (VWR). Non-specifically bound primaryantibody was removed by washing the blot for ten minutes with 1×PBScontaining 0.2% Tween 20. This was repeated four times. Secondaryantibody donkey anti-rabbit (Amersham) was incubated with the blot underthe same conditions as the primary antibody. Excess secondary antibodywas washed as described above. Blots were developed with the ECL WesternBlotting detection reagents according to the manufacturer'sinstructions. Blots were exposed to Medtec's Full Speed Blue Film(Medtec) or Amersham's ECL Hyperfilm (Amersham) for one second to fiveminutes. Film was developed in a KODAK Automatic Developer.

Determination of Lectin Binding Ability of Pro-Ricin Variant

An Immulon 2 plate (VWR) was coated with 100 μl per well of 10 μg/ml ofasialofetuin and left overnight at 4° C. The plate was washed with 3×300μl per well with ddH₂O using an automated plate washer (BioRad). Theplate was blocked for one hour at 37° C. by adding 300 μL per well ofPBS containing 1% ovalbumin. The plate was washed again as above.Proricin variant PAP-protein was added to the plate in various dilutionsin 1× Column Buffer, (50 mM Tris, 100 mM NaCl, pH 7.5). A standard curveof RCA₆₀ (Sigma) from 1-10 ng was also included. The plate was incubatedfor 1 h at 37° C. The plate was washed as above. Anti-ricin monoclonalantibody (Sigma) was diluted 1:3000 in 1×PBS containing 0.5% ovalburninand 0.1% Tween-20, added at 100 μL per well and incubated for 1 h at 37°C. The plate was washed as above. Donkey anti-rabbit polyclonal antibodywas diluted 1:3000 in 1×PBS containing 0.5% ovalburnin, 0.1% Tween-20,and added at 100 μL per well and incubated for 1 h at 37° C. The platewas given a final wash as described above. Substrate was added to plateat 100 μL per well (1 mg/mL o-phenylenediamine (in H₂O), 1 μL/mL H₂O₂)and after development 25 μL of stop solution (20% H₂SO₄) was added andthe absorbance read (A490 nm-A630 nm) using a SPECTRA MAX 340 platereader (Molecular Devices).

Determination of PAP-Protein Activity Using the Rabbit ReticulocyteAssay

Ricin samples were prepared for reduction.

-   A) RCA₆₀=3,500 ng/μL of RCA₆₀+997 μL 1× Endo buffer (25 mM Tris, 25    mM KCl, 5 mM MGCl₂, pH 7.6)    -   Reduction=95 μL of 10 ng/μL+5 μL β-mercaptoethanol-   B) Ricin variants    -   Reduction=40 μL variant+2 μL β-mercaptoethanol    -   The ricin standard and the variants were incubated for 30        minutes at room temperature.

Ricin—Rabbit Reticulocyte Lysate Reaction

The required number of 0.5 mL tubes were labelled. (2 25 tubes for eachsample, + and −aniline). To each of the sample tubes 20 μL of 1× endobuffer was added, and 30 μL of buffer was added to the controls. To thesample tubes either 10 μL of 10 ng/μL, Ricin or 10 μL of variant wasadded. Finally, 30 μL of rabbit reticulocyte lysate was added to all thetubes. The samples were incubated for 30 minutes at 30° C. using thethermal block. Samples were removed from the 0.5 mL tube and contentsadded into a 1.5 mL tube containing 1 mL of TRIZOL (Gibco). Samples wereincubated for 15 minutes at room temperature. After the incubation, 200μL of chloroform was added, and the sample was vortexed and spun at12,000 g for 15 minutes at 4° C. The top aqueous layer from the sampleswas removed and contents added to a 1 mL tube containing 500 μL ofisopropanol. Samples were incubated for 15 minutes at room temperatureand then centrifuged at 12,000 for 15 minutes at 4° C. Supernatant wasremoved and the pellets were washed with 1 mL of 70% ethanol.Centrifugation at 12,000 g for 5 minutes at 4° C. pelleted the RNA. Allbut approximately 20 μL of the supernatant was removed and the RNApellet was allowed to air dry. Pellets from the other samples (+anilinesamples) were dissolved in 20 μL of DEPC treated ddH₂O. An 80 μL aliquotof 1 M aniline (distilled) with 2.8 M acetic acid was added to these RNAsamples and transferred to a fresh 0.5 mL tube. The samples wereincubated in the dark for 3 minutes at 60° C. RNA was precipitated byadding 100 μl, of 95% ethanol and 5 μL of 3M sodium acetate, pH 5.2 toeach tube and centrifuging at 12,000 g for 30 minutes at 4° C. Pelletswere washed with 1 mL 70% ethanol and centrifuged again at 12,000 g for5 minutes at 4° C. to precipitate RNA. The supernatant was removed andair dried. These pellets were dissolved in 10 μL of 0.1× E buffer. Toall samples, 10 μL of formamide loading dye was added. The RNA ladder(BRL) (8 μL of ladder+8 μL of loading dye) was also included. Sampleswere incubated for 2 minutes at 70° C. on the thermal block.Electrophoresis was carried out on the samples using 1.2% agarose, 50%formamide gels in 0.1× E buffer+0.2% SDS. The gel was run for 90 minutesat 75 volts. RNA was visualized by staining the gel in 1 μg/mL ethidiumbromide in running buffer for 45 minutes. The gel was examined on a 302nm UV box, photographed using the gel documentation system and saved toa computer disk.

Results: Protein Expression Yields

Aliquots were taken at each stop of the harvesting/purification andtested. Yields of functional ricin variant were determined by ELISA.Typical results of an 3400 mL prep of infected T ni cells are givenbelow.

Aliquot μg PAP 304 Before concentration and dialysis 14,472 afterconcentration and dialysis 13,611 alpha-Lactose agarose column flowthrough 418 alpha-Lactose agarose column elution 8,682

Yield: 8,682/14,472=60% Purification of PAP-Protein and Western Analysisof Column Fractions

Partially purified PAP-protein was applied to Superdex 75 and 200(16/60) columns connected in series in order to remove the contaminatingnon-specifically processed PAP-protein. Eluted fractions were tested viaWestern analysis as described above and the fractions containing themost pure protein were pooled, concentrated and dialyzed against 1×PBSbuffer and then sterilized by filtration (Millipore). Final purifiedPAP-protein has less than 1% processed variant.

The purified PAP-protein was tested for susceptibility to cleavage bythe particular protease and for activation of the A chain of theproricin variant, (inhibition of protein synthesis). Typically,PAP-protein was incubated with and without protease for a specified timeperiod and then electrophoresed and blotted. Cleaved PAP-protein willrun as two 30 kDa proteins (B is slightly larger) under reducing(SDS-PAGE) conditions. Unprocessed PAP-protein, which contains thelinker region, will migrate at 60 kDa.

Activation of PAP-Protein Variant with Specific Protease

Activation of protease treated PAP-protein is based on the method of Mayet al. (EMBO Journal. 8 301-8, 1989). Activation of ricin A chain uponcleavage of the intermediary linker results in catalytic depurination ofthe adenosine 4325 residue of 28S or 26S rRNA. This depurination rendersthe molecule susceptible to amine-catalyzed hydrolysis by aniline of thephosphodiester bond on either side of the modification site. The resultis a diagnostic 390 base band. As such, reticulocyte ribosomes incubatedwith biochemically purified ricin A chain, released the characteristicRNA fragment upon aniline treatment of isolated rRNA (May, M. J. et al.Embo. Journal, 8:301-308 at 302-303 (1989)). It is on this basis thatthe assay allows for the determination of activity of a ricin A chainwhich has been cleaved from the intact unit containing a particularvariant linker sequence.

Example 3 In Vitro Protease Digestion of Proricin Variants:

Affinity-purified proricin variant is treated with individualdisease-specific proteases to confirm specific cleavage in the linkerregion. Ricin-like toxin variants are eluted from the lactose-agarosematrix in protease digestion buffer (50 mM NaCl, 50 mM Na-acetate, pH5.5, 1 mM dithiothreitol) containing 100 mM lactose. Proricin substrateis then incubated at 37° C. for 60 minutes with a disease-specificprotease. The cleavage products consisting ricin A and B chains areidentified using SDS/PAGE (Sambrook et al., Molecular Cloning: aLaboratory Manual, 2nd. ed., Cold Spring Harbor Press, 1989), followedby Western blot analysis using anti-ricin antibodies (Sigma). FIG. 19shows the cleavage products of an MMP-9 digestion of PAP323, PAP324 andPAP325.

Matrix metalloproteinases may be prepared substantially as described byLark, M. W. et al. (Proceedings of the 4th International Conference ofthe Inflammation Research Association Abstract 145 (1988)) and Welch, A.R. et al. (Arch. Biochem. Biophys. 324:59-64 (1995)).

Urokinase plasminogen activator may be prepared substantially asdescribed by Holmberg, L. et al. (Biochim Biophys Acta, 445:215-222,(1976)) and Someno, T. et al. (J Biochem 97:1493-1500 (1985)).

Example 4 Cytotoxicity of Ricin and Ricin Variants on Cell Lines CellLines COS-I (African Green Monkey Kidney Cells)

This is an SV40 transformed cell line which was prepared fromestablished simian cells CV-1. (Reference: Gluzman, Y. (1975) Cell, 23,175-182)(ATCC CRL 1650).

HT-1080 Human Fibrosarcoma

(ATCC CCL 121) This cell line was shown to produce active MMP-9 intissue culture. (References: Moore et al. (1997) Gynecologic Oncology65, 83-88.)

Cell Preparation

After washing with 1×PBS (0.137 M NaCl, 2.68 mM KCl, 8.10 mM Na₂HP0₄,1.47 mM KH₂PO₄), cells in log phase growth were removed from plates with1× trypsin/EDTA (Gibco/BRL). The cells were centrifuged at 1100 rpm for3 min, resuspended in Dulbecco's Modified Eagle Medium containing 10%FBS and 1× pen/strep, and then counted using a haemocytometer. They wereadjusted to a concentration of 5×10⁴ cells·ml⁻¹. One hundred microlitersper well of cells was added to wells 2B-2G through to wells 9B-9G of aFalcon 96 well tissue culture plate. A separate 96 well tissue cultureplate was used for each sample of Ricin or Ricin variant. The plateswere incubated at 37° C. with 5% CO₂ for 24 hours.

Toxin Preparation

The Ricin and Ricin variants were sterile filtered using a 0.22 μmfilter (Millipore). The concentration of the sterile samples were thenquantified by A₂₈₀ and confirmed by BCA measurements (Pierce). For thevariants digested with the MMP-9 protease in vitro, the digests werecarried out as described in the digestion procedure for each protease.The digests were then diluted in the 1000 ng·ml⁻¹ dilution and sterilefiltered. Ricin and Ricin variants were serially diluted to thefollowing concentrations: 1000 ng·ml⁻¹, 100 ng·ml⁻¹, 10 ng·ml⁻¹, 1ng·ml⁻¹, 0.1 ng·ml⁻¹, 0.01 ng·ml⁻¹, 0.001 ng·ml⁻¹ with media containing10% FBS and 1× pen/strep.

Application of Toxin or Variants to Plates

Columns 2 to 9 were labeled: control, 1000 ng·ml⁻¹, 100 ng·ml⁻¹, 10ng·ml⁻¹, 1 ng·ml⁻¹, 0.1 ng·ml⁻¹, 0.01 ng·ml⁻¹, 0.001 ng·ml⁻¹consecutively. The media was removed from all the sample wells with amultichannel pipettor. For each plate of variant and toxin, 50 μl ofmedia was added to wells 2B to 2G as the control, and 50 μl of eachsample dilution was added to the corresponding columns. The plates wereincubated for one hour at 37° C. with 5% CO₂, then washed once andreplaced with media, then incubated for 48 hours at 37° C. with 5% CO₂.

Sample Application

The whole amount of media (and/or toxin) was removed from each well witha multichannel pipettor, and replaced with 100 μl of the substratemixture (Promega Cell Titer 96 Aqueous Non-Radioactive CellProliferation Assay Kit). The plates were incubated at 37° C. with 5%CO₂ for 2 to 4 hours, and subsequently read with a Spectramax 340 96well plate reader at 490 nm. The IC₅₀ values were calculated using theGRAFIT software program.

Results

The results of the cytotoxicity assay are shown in Tables 1 to 4. Inalmost all cases the novel variants show preferential activation in thetumour cell line HT-1080 (human fibrosarcoma) as compared with thenon-tumourogenic cell line COS-1 (immortalized cell line form the kidneyof an African green monkey).

Example 5 Maximum Tolerable Dose Data

The protocol for the maximum tolerable dose (MTD) study involved threeintravenous injections of variant, on days 1, 5 and 9, into the tailvein of either a Nude/SCID mouse. Three animals were used for each dosetested. The samples were diluted into saline solution containing 100μg/mL Bovine Serum Albumin on the same day as the injection. Animalswere observed for 14 days after dosing. Any surviving animals wereeuthanized after 14 days of study. The MTD value was defined as thehighest dose of sample tested where all animals in the group survived.The results are presented in Table 5.

These results demonstrate that linkers of the invention in proricinvariants decrease the toxicity of the recombinant proteins.

Example 6

In vivo Studies

(a) Protocol for A431 Animal Model Studies

Tumour growth was monitored daily by measuring tumour dimensions withcalipers. The treatment initiation date was dependent on the rate oftumour growth. Four groups (4 mice per group) of mice develop tumours ofthe desired size (50 mm³-100 mm³) Such mice are weighed and treatmentinitiated. This treatment initiation date is considered as day 1, andthe mice were given a bolus intravenous injection of variant on thisday. Injections were administered through the lateral tail vein. Thetreatment groups are shown in Table 6.

All samples and buffer were made up in saline solution containing 100μg/mL Bovine Serum Albumin.

(b) In Vivo Efficacy Studies

Subcutaneous A431 tumours were established in SCID mice. The tumourswere treated with either PAP304 or PAP305 when the tumours reached 50mm³ on Days 1, 5 and 9. The results shown in FIGS. 20 and 21 demonstratethat the linker decreases the toxicity of the variant (as compared withricin) and the variants PAP304 and PAP305 are activated at or near theA431 (human epithelial carcinoma) solid tumour in mice. A very excitingresult is shown in FIG. 20. In this study, the variant PAP304 was ableto slow down the growth of A431 solid tumour (17 day delay), without anysigns of dose limiting toxicity (e.g., no weight loss or death).

(c) Protocol and Efficacy for Testing PAP304 against P388 MurineLeukemia Tumour Model

Mice were grouped according to body weight. Animals (n=4) wereinoculated (Day=0) with 1×106 cells implanted subcutaneously in theflank of the BDF-1 mouse in a volume of 50 μL with a 28 g needle. P388murine leukemia cells from the ATCC tumor repository were maintained asan ascitic fluid in the BDF-1 mouse which were passaged to new miceweekly. The cells used for experiment were used within passage 3-20. Forthe experiment, cells were rinsed with Hanks Balanced Salt Solution,counted on a heamocytometer and diluted with HBSS to a concentration of20×10⁶ cells/ml. PAP304 was injected intravenously on days 3, 6 and 9after tumour injection. The results are shown in FIG. 22. A significantdelay in tumor growth in the murine tumor model.

Having illustrated and described the principles of the invention in apreferred embodiment, it should be appreciated to those skilled in theart that the invention can be modified in arrangement and detail withoutdeparture from such principles. We claim all modifications coming withinthe scope of the following claims.

All publications, patents and patent applications referred to herein areincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

FULL CITATIONS FOR CERTAIN REFERENCES REFERRED TO IN THE SPECIFICATION

-   Bever Jr., C. T., Panitch, H. S., and Johnson, K. P. (1994)    Neurology 44(4), 745-8. increased cathepsin B activity in peripheral    blood mononuclear 5 cells of multiple sclerosis patients.-   Cohen, P., Graves, H. C., Peehl, D. M., Kamarei, M., Giudice, L. C.,    and Rosenfeld, R. G. (1992) journal of Clinal Endocrinology and    Metabolism 75(4), 1046-53. Prostate-specific antigen (PSA) is an    insulin-like growth factor binding protein-3 protease found in    seminal plasma.-   Conover, C. A. and De Leon, D. D. (1994) J. Biol. Chem. 269(10),    7076-80. Acid activated insulin-like growth factor-binding protein-3    proteolysis in normal and transformed cells. Role of cathepsin D.-   Cuzner, M. L., Opdenakker, G. Plasminogen activators and matrix    metalloproteases, mediators of extracellular proteolysis in    inflammatory demyelination of the central nervous system. J.    Neuroimmunol 94(1-2):1-14 (1999).-   Desreumaux, P., Huet, G., Zerimech, F., Gambiez, L., Balduyck, M.,    Baron, P., Degand, P., Cortot, A., Colombel, J. F., Janin, A. Acute    inflammatory intestinal vascular lesions and in situ abnormalities    of the plasminogen activation system in Crohn's disease. Eur. J.    Gastroenterol Hepatol, 11(10): 1113-9 (1999).-   Hansen, G., Schuster, A., Zubrod, C., and Wahn, V. (1995) Resp    62(3), 117-24. Alpha 1-proteinase inhibitor abrogates proteolytic    and secretagogue activity of cystic fibrosis sputum.-   Muller, H. L., Oh, Y., Gargosky, S. E., Lehrnbecher, T., Hintz, R.    L., and Rosenfeld, R. G. (1993) journal of Clinical Endocrinology    and Metabolism 77(5), 1113-9. Concentrations of insulin-like growth    factor (IGF)-binding protein-3 (IGFBP-3), IGF, and 1GFBP-3 protease    activity in cerebrospinal fluid of children with leukemia, central    nervous system tumor, or meningitis.-   Pap, G., Eberhardt, R., Rocken, C., Nebelung, W., Neumann, H. W.,    Roessner, A. Expression of stromelysin and urokinase type    plasminogen activator protein in resection specimens and biopsies at    different stages of osteoarthritis of the knee. Pathol Res Pract    196(4):219-26 (2000).-   Slot, O., Brunner, N., Locht, H., Oxholm, P., Stephens, R. W.,    Soluble urokinase plasminogen activator receptor in plasma of    patients with inflammatory rheumatic disorders: increased    concentrations in rheumatoid arthritis. Ann Rheum Dis, 58(8):488-92    (1999).

1) Cytotoxicity of Selected Variants

TABLE 1 Selected Variants against COS-1 Cells - Target Protease MMP-9Ricin PAP220 PAP301 PAP302 PAP303 PAP304 PAP305 PAP308 Linker Length —23 23 16 15 8 12 12 (residues) Reduction in 1X 23X 24X 118X 63X 1220X145X 89X toxicity relative to Ricin

TABLE 2 Selected Variants against HT1080 Cells - Target Protease MMP-9Ricin PAP220 PAP301 PAP302 PAP303 PAP304 PAP305 PAP308 Linker Length —23 23 16 15 8 12 12 (residues) Reduction in 1X 4X 5X 24X 12X 137X 38X21X toxicity relative to Ricin2) Cytotoxicity Data from Selected Variants

TABLE 3 Selected Variants against COS-1 cells MMP9 Variants Ricin PAP316PAP318 PAP323 PAP324 PAP325 Linker Length — 23 23 21 19 17 (residues)Reduction in 1X 39X 100X 65X 67X 82X toxicity relative to Ricin UPAVariants Ricin PAP313 PAP314 PAP315 PAP320 PAP321 PAP322 Linker Length —7 15 14 13 11 9 (residues) Reduction in 1X 110X 52X 75X 55X 1283X 82Xtoxicity relative to Ricin

TABLE 4 Selected Variants against HT1080 Cells MMP9 Variants RicinPAP316 PAP318 PAP323 PAP324 PAP325 Linker Length — 23 23 21 19 17(residues) Reduction in 1X 13X 51X 15X 14X 20X toxicity relative toRicin UPA Variants Ricin PAP313 PAP314 PAP315 PAP320 PAP321 PAP322Linker Length — 7 15 14 13 11 9 (residues) Reduction in 1X 43X 27X 18X14X 367X 51X toxicity relative to Ricin

TABLE 5 Maximum Tolerable Dose of MMP9 Variants MMP9 Variant Linker SizeIn Vivo (μg/kg) PAP301 23 8 PAP302 16 40 PAP303 15 10 PAP304 8 150PAP305 12 20 PAP308 12 30 PAP309 23 20 PAP316 23 20 PAP318 23 <20 PAP32321 15 PAP324 19 20 PAP325 17 20 (cf. Ricin - 1.6 μg/kg and PAP220 - 13μg/kg)

TABLE 6 Drug Dose Treatment Group Sample (μg/kg) (days) 1 Control -Buffer 0 1, 5, and 9 2 PAP304 75 1, 5, and 9 3 PAP304 100 1, 5, and 9 4PAP304 150 1, 5, and 9

1-42. (canceled)
 43. A purified and isolated nucleic acid moleculecomprising (a) a nucleotide sequence encoding an A chain of a ricin-liketoxin, (b) a nucleotide sequence encoding a B chain of a ricin-liketoxin and (c) a nucleotide sequence encoding a heterologous linker aminoacid sequence linking the A and B chains, the heterologous linkersequence containing a cleavage recognition site for a specific protease.44. A nucleic acid molecule of claim 43 wherein the specific protease isan MMP or UPA.
 45. A nucleic acid molecule according to claim 43 whereinthe protease is associated with a cancer cell.
 46. A nucleic acidmolecule according to claim 45 wherein the cancer cell is one found inT- and B-cell lymphoproliferative diseases, ovarian cancer, pancreaticcancer, head and neck cancer, squamous cell carcinoma, gastrointestinalcancer, breast cancer, prostate, cancer or non small cell lung cancer.47. A nucleic acid molecule according to claim 43 wherein the proteaseis associated with an inflammatory cell.
 48. A nucleic acid moleculeaccording to claim 47 wherein the cell is one found in rheumatoidarthritis, atherosclerotic cells, Crohn's disease, or central nervoussystem disease.
 49. A nucleic acid molecule of claim 43 wherein the Achain is ricin A chain, abrin toxin A chain, diphtheria toxin A chain,Domain III of Pseudomonas exotoxin, volkensin toxin A chain, choleratoxin A chain, modeccin toxin A chain, viscumin toxin A chain or shigatoxin A chain.
 50. A nucleic acid molecule of claim 43 wherein the Bchain is ricin B chain, abrin toxin B chain, diphtheria toxin B chain,Domain I/II of Pseudomonas exotoxin, volkensin toxin B chain, choleratoxin B chain, modeccin toxin B chain, viscumin toxin B chain or shigatoxin B chain.
 51. A nucleic acid molecule according to claim 43 havinga nucleic acid sequence selected from the group consisting of thenucleic acid sequence of pAP301 as shown in FIG. 1B (SEQ ID NO:5); thenucleic acid sequence of pAP302 as shown in FIG. 2B (SEQ ID NO:12); thenucleic acid sequence of pAP303 as shown in FIG. 3B (SEQ ID NO:19); thenucleic acid sequence of pAP304 as shown in FIG. 4B (SEQ ID NO:26); thenucleic acid sequence of pAP305 as shown in FIG. 5B (SEQ ID NO:33); thenucleic acid 5 sequence of pAP308 as shown in FIG. 6B (SEQ ID NO:40);the nucleic acid sequence of pAP309 as shown in FIG. 7B (SEQ ID NO:47);the nucleic acid sequence of pAP313 as shown in FIG. 8B (SEQ ID NO:54);the nucleic acid sequence of pAP314 as shown in FIG. 9B (SEQ ID NO:61);the nucleic acid sequence of pAP315 as shown in FIG. 10B (SEQ ID NO:68);the nucleic acid sequence of pAP316 as shown in FIG. 11B (SEQ ID NO:75);the nucleic acid sequence of pAP318 as shown in FIG. 12B (SEQ ID NO:82);the nucleic acid sequence of pAP320 as shown in FIG. 13B (SEQ ID NO:89);the nucleic acid sequence of pAP321 as shown in FIG. 14B (SEQ ID NO:96);the nucleic acid sequence of pAP322 as shown in FIG. 15B (SEQ IDNO:103); the nucleic acid sequence of pAP323 as shown in FIG. 16B (SEQID NO:110); the nucleic acid sequence of pAP324 as shown in FIG. 17B(SEQ ID NO:117); and the nucleic acid sequence of pAP325 as shown inFIG. 18B (SEQ ID NO:124).
 52. A nucleic acid molecule according to claim43 wherein the nucleotide sequence of the linker is selected from thegroup consisting of: the nucleic acid sequence of pAP301 as shown inFIG. 1A (SEQ ID NO:4); the nucleic acid sequence of pAP302 as shown inFIG. 2A (SEQ ID NO:11); the nucleic acid sequence of pAP303 as shown inFIG. 3A (SEQ ID NO:18); the nucleic acid sequence of pAP304 as shown inFIG. 4A (SEQ ID NO:25); the nucleic acid sequence of pAP305 as shown inFIG. 5A (SEQ ID NO:32); the nucleic acid sequence of pAP308 as shown inFIG. 6A (SEQ ID NO:39); the nucleic acid sequence of pAP309 as shown inFIG. 7A (SEQ ID NO:46); the nucleic acid sequence of pAP313 as shown inFIG. 8A (SEQ ID NO:53); the nucleic acid sequence of pAP314 as shown inFIG. 9A (SEQ ID NO:60); the nucleic acid sequence of pAP315 as shown inFIG. 10A (SEQ ID NO:67); the nucleic acid sequence of pAP316 as shown inFIG. 11A (SEQ ID NO:74); the nucleic acid sequence of pAP318 as shown inFIG. 12A (SEQ ID NO:81); the nucleic acid sequence of pAP320 as shown inFIG. 13A (SEQ ID NO:88); the nucleic acid sequence of pAP321 as shown inFIG. 14A (SEQ ID NO:95); the nucleic acid sequence of pAP322 as shown inFIG. 15A (SEQ ID NO:102); the nucleic acid sequence of pAP323 as shownin FIG. 16A (SEQ ID NO:109); the nucleic acid sequence of pAP324 asshown in FIG. 17A (SEQ ID NO:116); and the nucleic acid sequence ofpAP325 as shown in FIG. 18A (SEQ ID NO:123).
 53. A plasmid incorporatingthe nucleic acid molecule of claim
 43. 54. A baculovirus transfer vectorincorporating the nucleic acid molecule according to claim
 43. 55. Arecombinant protein comprising an A chain of a ricin-like toxin, a Bchain of a ricin-like toxin and a heterologous linker amino acidsequence, linking the A and B chains, wherein the linker sequencecontains a cleavage recognition site for a specific protease.
 56. Arecombinant protein of claim 55 wherein the specific protease is an MMPor UPA.
 57. A protein according to claim 55 wherein the protease isassociated with a cancer cell.
 58. A protein according to claim 57wherein the cancer cell is one found in T- and B celllymphoproliferative diseases, ovarian cancer, pancreatic cancer, headand neck cancer, squamous cell carcinoma, gastrointestinal cancer,breast cancer, prostate, cancer or non small cell lung cancer.
 59. Aprotein according to claim 55 wherein the protease is associated with aninflammatory cell.
 60. A protein according to claim 59 wherein the cellis one found in rheumatoid arthritis, atherosclerotic cells, Crohn'sdisease, or central nervous system disease.
 61. A recombinant protein ofclaim 55 wherein the A chain is ricin A chain, abrin toxin A chain,diphtheria toxin A chain, Domain III of Pseudomonas exotoxin, volkensintoxin A chain, cholera toxin A chain, modeccin toxin A chain, viscumintoxin A chain, or shiga toxin A chain.
 62. A recombinant protein ofclaim 55 wherein the B chain is ricin B chain, abrin toxin B chain,diphtheria toxin B chain, Domain I/II of Pseudomonas exotoxin, volkensintoxin B chain, cholera toxin B chain, modeccin toxin B chain, viscumintoxin B chain, or shiga toxin B chain.
 63. A recombinant protein ofclaim 55 wherein the linker amino acid sequence is selected from thegroup consisting of: the amino acid sequence of PAP301 as shown in FIG.1C (SEQ ID NO:7); the amino acid sequence of PAP309 as shown in FIG. 7C(SEQ ID NO:49); the amino acid sequence of PAP314 as shown in FIG. 9C(SEQ ID NO:63); the amino acid sequence of PAP315 as shown in FIG. 10C(SEQ ID NO:70); the amino acid sequence of PAP318 as shown in FIG. 12C(SEQ ID NO:84); the amino acid sequence of PAP320 as shown in FIG. 13C(SEQ ID NO:91); the amino acid sequence of PAP321 as shown in FIG. 14C(SEQ ID NO:98); and the amino acid sequence of PAP322 as shown in FIG.15C (SEQ ID NO:105.
 64. A method of inhibiting or destroying cellshaving a specific protease comprising the steps of: (a) preparing apurified and isolated nucleic acid having a nucleotide sequence encodingan A chain of a ricin-like toxin, a B chain of a ricin-like toxin, and aheterologous linker amino acid sequence, linking the A and B chains,wherein the linker sequence contains a cleavage recognition site for theprotease; (b) introducing the nucleic acid into a host cell andexpressing the nucleic acid in the host cell to obtain a recombinantprotein comprising an A chain of a ricin-like toxin, a B chain of aricin-like toxin and a linker amino acid sequence; (c) suspending theprotein in a pharmaceutically acceptable carrier, diluent or excipient,and (d) contacting the cells with the recombinant protein.
 65. A methodaccording to claim 64 wherein the protease is an MMP or UPA.
 66. Amethod according to claim 64 wherein the protease is associated with acancer cell.
 67. A method according to claim 66 wherein the cancer cellis one found in T- and B cell lymphoproliferative diseases, ovariancancer, pancreatic cancer, head and neck cancer, squamous cellcarcinoma, gastrointestinal cancer, breast cancer, prostate, cancer ornon small cell lung cancer.
 68. A method according to claim 64 whereinthe protease is associated with an inflammatory cell.
 69. A methodaccording to claim 68 wherein the cell is one found in rheumatoidarthritis, atherosclerotic cells, Crohn's disease, or central nervoussystem disease.
 70. A method of inhibiting or destroying cells having aspecific protease comprising contacting the cells with an effectiveamount a recombinant protein according to claim
 55. 71. A method oftreating a cell having a specific protease comprising administering aneffective amount of a recombinant protein according to claim 55 to ananimal in need thereof.
 72. A method of treating a cell having aspecific protease comprising administering an effective amount of anucleic acid molecule according to claim 43 to an animal in needthereof.
 73. A process for preparing a pharmaceutical for treating acell having a specific protease comprising the steps of: (a) preparing apurified and isolated nucleic acid having a nucleotide sequence encodingan A chain of a ricin-like toxin, a B chain of a ricin-like toxin, and aheterologous linker amino acid sequence, linking the A and B chains,wherein the linker sequence contains a cleavage recognition site for aspecific protease; (b) introducing the nucleic acid into a host cell andexpressing the nucleic acid in the host cell to obtain a recombinantprotein comprising an A chain of a ricin-like toxin, a B chain of aricin-like toxin and a linker amino acid sequence; (c) suspending theprotein in a pharmaceutically acceptable carrier, diluent or excipient.74. A process according to claim 73 wherein the protease is an MMP orUPA.
 75. A process according to claim 73 wherein the protease isassociated with a cancer cell.
 76. A process according to claim 75wherein the cancer cell is one found in T- and B celllymphoproliferative diseases, ovarian cancer, pancreatic cancer, headand neck cancer, squamous cell carcinoma, gastrointestinal cancer,breast cancer, prostate, cancer or non small cell lung cancer.
 77. Aprocess according to claim 73 wherein the protease is associated with aninflammatory cell.
 78. A process according to claim 77 wherein the cellis one found in rheumatoid arthritis, atherosclerotic cells, Crohn'sdisease, or central nervous system disease.
 79. A pharmaceuticalcomposition for treating cancer comprising a recombinant protein ofclaim 55 and a pharmaceutically acceptable carrier, diluent orexcipient.
 80. A pharmaceutical composition for treating inflammationcomprising a recombinant protein of claim 55 and a pharmaceuticallyacceptable carrier, diluent or excipient.
 81. A pharmaceuticalcomposition for treating a cell having a specific protease comprising anucleic acid molecule of claim 43 and a pharmaceutically acceptablecarrier, diluent or excipient.
 82. A pharmaceutical composition fortreating a cell having a specific protease comprising an amino acidmolecule of claim 43 and a pharmaceutically acceptable carrier, diluentor excipient.
 83. A purified and isolated nucleic acid molecule having anucleic acid sequence selected from the group consisting of: the nucleicacid sequence of pAP301 as shown in FIG. 1A (SEQ ID NO:4); the nucleicacid sequence of pAP302 as shown in FIG. 2A (SEQ ID NO:11); the nucleicacid sequence of pAP303 as shown in FIG. 3A (SEQ ID NO:18); the nucleicacid sequence of pAP304 as shown in FIG. 4A (SEQ ID NO:25); the nucleicacid sequence of pAP305 as shown in FIG. 5A (SEQ ID NO:32); the nucleicacid sequence of pAP308 as shown in FIG. 6A (SEQ ID NO:39); the nucleicacid sequence of pAP309 as shown in FIG. 7A (SEQ ID NO:46); the nucleicacid sequence of pAP313 as shown in FIG. 8A (SEQ ID NO:53); the nucleicacid sequence of pAP314 as shown in FIG. 9A (SEQ ID NO:60); the nucleicacid sequence of pAP315 as shown in FIG. 10A (SEQ ID NO:67); the nucleicacid sequence of pAP316 as shown in FIG. 11A (SEQ ID NO:74); the nucleicacid sequence of pAP318 as shown in FIG. 12A (SEQ ID NO:81); the nucleicacid sequence of pAP320 as shown in FIG. 13A (SEQ ID NO:88); the nucleicacid sequence of pAP321 as shown in FIG. 14A (SEQ ID NO:95); the nucleicacid sequence of pAP322 as shown in FIG. 15A (SEQ ID NO:102); thenucleic acid sequence of pAP323 as shown in FIG. 16A (SEQ ID NO:109);the nucleic acid sequence of pAP324 as shown in FIG. 17A (SEQ IDNO:116); and the nucleic acid sequence of pAP325 as shown in FIG. 18A(SEQ ID NO:123).
 84. A linker protein having an amino acid sequenceselected from the group consisting of: the amino acid sequence of PAP301as shown in FIG. 1C (SEQ ID NO:7); the amino acid sequence of PAP309 asshown in FIG. 7C (SEQ ID NO:49); the amino acid sequence of PAP314 asshown in FIG. 9C (SEQ ID NO:63); the amino acid sequence of PAP315 asshown in FIG. 10C (SEQ ID NO:70); the amino acid sequence of PAP318 asshown in FIG. 12C (SEQ ID NO:84); the amino acid sequence of PAP320 asshown in FIG. 13C (SEQ ID NO:91); the amino acid sequence of PAP321 asshown in FIG. 14C (SEQ ID NO:98); and the amino acid sequence of PAP322as shown in FIG. 15C (SEQ ID NO:105).